JP7157089B2 - Diagnosis and treatment of autoimmune diseases - Google Patents

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to the filing date of U.S. Provisional Patent Application No. 61/893,542, filed October 21, 2013, the entirety of which is incorporated herein by reference. incorporated into the book.

Plasma kallikrein (pKal) is the major bradykinin-producing enzyme in the circulatory system and a component of the plasma kallikrein-kinin system (KKS) (Colman, RW and Schmaier, AH (1997) Blood 90 , 3819-3843). Activation of pKal occurs through the contact system, which has been demonstrated to be responsible for the disease pathology associated with hereditary angioedema (HAE) (Zuraw, BL and Christiansen, S.; C. (2008) Expert Opin Investig Drugs 17, 697-706). Bradykinin is an important mediator of pain, inflammation, edema and angiogenesis (Maurer, M. et al. (2011) Allergy 66, 1397-1406; Colman, RW (2006) Curr Pharm Des 12, 2599-2607) .

SUMMARY The present disclosure is the finding that the concentration of truncated high molecular weight kininogen (HMWK) is elevated in patients with autoimmune diseases such as rheumatoid arthritis (RA), Crohn's disease (CD) and ulcerative colitis (UC). based on. Thus, provided herein are methods for diagnosing an autoimmune disease such as RA, CD or UC, monitoring the progression of an autoimmune disease, or determining the efficacy of a treatment for an autoimmune disease based on the concentration of truncated HMWK. is disclosed.

In one aspect, the disclosure provides a method of diagnosing an autoimmune disease (e.g., RA, CD or UC) in a subject, wherein the method comprises: (i) a subject suspected of having an autoimmune disease; (e.g., a serum or plasma sample); (ii) measuring the concentration of truncated high molecular weight kininogen (HMWK) in said biological sample; and (iii) identifying that the subject has or is at risk for an autoimmune disease when the concentration of truncated HMWK is elevated compared to a control sample.

In some embodiments, the concentration of truncated HMWK is measured by an assay that includes a binding agent (eg, an antibody) that is specific for truncated HMWK. The assay can be an enzyme-linked immunosorbent assay (ELISA) or an immunoblot assay, such as a Western blot assay with LiCor detection.

The method can further comprise administering a treatment for the autoimmune disease to the subject. In certain embodiments, the subject is administered an effective amount of a plasma kallikrein (pKal) inhibitor, such as those described herein.

In another aspect, the disclosure provides a method of monitoring the progression of an autoimmune disease (e.g., RA, CD or UC) in a subject, the method comprising: (i) having the autoimmune disease at a first time point; providing a first biological sample of a subject suspected of being infected; (ii) measuring a first concentration of high molecular weight kininogen (HMWK) in said first biological sample; iii) providing a second biological sample of said subject at a second time point subsequent to said first time point; (iv) a second concentration of truncated HMWK in said second biological sample. and (v) determining the progression of the autoimmune disease in the subject based on the change in concentration of truncated HMWK in the first and second biological samples. If the second concentration of truncated HMWK is higher than the first concentration of truncated HMWK, then the autoimmune disease is progressing in the subject, or the subject has developed or has developed an autoimmune disease. indicates that there is a risk of

In some embodiments, the first biological sample, the second biological sample, or both are serum or plasma samples. In other embodiments, the first or second concentration of truncated HMWK is determined by an assay that includes a binding agent (eg, antibody) specific for truncated HMWK. In some examples, the assay may be an enzyme-linked immunosorbent assay (ELISA) or an immunoblot assay, such as a Western blot assay, including LiCor detection.

The method can further comprise administering a treatment for the autoimmune disease to the subject. In certain embodiments, the subject is administered an effective amount of a plasma kallikrein (pKal) inhibitor, such as those described herein.

Further, the present disclosure provides a method of determining efficacy of treatment for an autoimmune disease (e.g., RA, CD or UC) in a patient, the method comprising: (i) a patient undergoing treatment for an autoimmune disease in the course of treatment; (ii) measuring the concentration of high molecular weight kininogen (HMWK) in the plurality of biological samples; and (iii) Determining efficacy of treatment in the patient based on changes in concentration of truncated HMWK over the course of treatment. A decrease in the concentration of truncated HMWK during the course of treatment indicates that the treatment is effective in the patient.

In certain embodiments, the treatment includes at least one plasma kallikrein inhibitor, such as those described herein. In other embodiments, the concentration of truncated HMWK in a plurality of biological samples is measured by an assay comprising a binding agent (eg, antibody) specific for truncated HMWK. In some examples, the assay is an enzyme-linked immunosorbent assay (ELISA) or an immunoblot assay, such as a Western blot assay, including LiCor detection. In any of the methods described herein, the biological sample used therein can contain a protease inhibitor or protease inhibitor cocktail that is added to the biological sample after collection.

A kallikrein inhibitor useful in the method can be, for example, a plasma kallikrein (pKal) inhibitor. In some embodiments, the inhibitor is a plasma kallikrein inhibitor.

Kallikrein inhibitors useful in the methods include Kunitz domain polypeptides known in the art or described herein, any such Kunitz domain containing larger polypeptides (kallikrein inhibitor polypeptides), provided that it binds and inhibits kallikrein as measured in a standard assay), a kallikrein-binding protein (e.g., an antibody such as an anti-plasma kallikrein antibody), or as described herein any of the other kallikrein inhibitors described herein.

ｐＫａｌ活性を阻害できる例示的なＫｕｎｉｔｚドメインペプチドは：Ｇｌｕ Ａｌａ Ｍｅｔ Ｈｉｓ Ｓｅｒ Ｐｈｅ Ｃｙｓ Ａｌａ Ｐｈｅ Ｌｙｓ Ａｌａ Ａｓｐ Ａｓｐ Ｇｌｙ Ｐｒｏ Ｃｙｓ Ａｒｇ Ａｌａ Ａｌａ Ｈｉｓ Ｐｒｏ Ａｒｇ Ｔｒｐ Ｐｈｅ Ｐｈｅ Ａｓｎ Ｉｌｅ Ｐｈｅ Ｔｈｒ Ａｒｇ Ｇｌｎ Ｃｙｓ Ｇｌｕ Ｇｌｕ Ｐｈｅ Ｉｌｅ Ｔｙｒ Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp (SEQ ID NO:2; SX-88), or a fragment thereof such as amino acids 3-60 of SEQ ID NO:2 including.

In certain embodiments, the kallikrein inhibitor comprises or consists of the framework region of the Kunitz domain and the first and second binding loop regions of the DX-88 polypeptide.

In some embodiments, the kallikrein inhibitor comprises or consists of a DX-88 polypeptide having about the 58 amino acid sequence of amino acids 3-60 of SEQ ID NO:2 or the 60 amino acid sequence of SEQ ID NO:2.

In certain embodiments, the kallikrein inhibitor comprises a plasma kallikrein-binding protein (eg, an antibody, such as an anti-plasma kallikrein antibody described herein).

In certain embodiments, a binding protein (e.g., an antibody such as a human antibody) binds to the same epitope as a protein described herein or competes for binding with a protein described herein. .

In some embodiments, the proteins described herein are M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to herein as DX-2922), X81-B01, X67-D03, X67-G04, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X124- G01 (also referred to herein as DX-2930), X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04. Such binding proteins are described, for example, in PCT Publication WO2012/094587 and US Patent Application Publication US2010183625, both of which are hereby incorporated by reference in their entirety.

In certain embodiments, the plasma kallikrein binding protein competes with or has the same epitope as X81-B01, X67-D03, X101-A01, M162-A04, X115-F02, X124-G01 or X63-G06 .

In some embodiments, the plasma kallikrein binding protein does not bind prekallikrein (eg, human prekallikrein), but binds the active form of plasma kallikrein (eg, human plasma kallikrein).

In certain embodiments, the protein binds at or near the active site of the catalytic domain of plasma kallikrein or a fragment thereof, or binds to an epitope overlapping the active site of plasma kallikrein.

In certain embodiments, the protein binds to one or more amino acids that form the catalytic triad of plasma kallikrein: His434, Asp483 and/or Ser578 (numbering is based on the human sequence). In other embodiments, the protein binds to one or more amino acids of Ser479, Tyr563 and/or Asp58 (numbering is based on the human sequence). In yet other embodiments, the plasma kallikrein binding protein binds to one or more amino acids of Arg551, Gln553, Tyr555 and/or Arg560 (amino acid position numbering is based on the human kallikrein sequence). In still other embodiments, the plasma kallikrein binding protein binds to one or more amino acids of Ser478, Asn481, Ser525 and/or Lys526 (amino acid position numbering is based on the human kallikrein sequence).

In certain embodiments, the plasma kallikrein-binding protein increases the production of factor XIIa and/or bradykinin, e.g. about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about greater than 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95%.

In certain embodiments, the plasma kallikrein-binding protein has an apparent inhibition constant (K _i,app ) of less than 1000 nM, 500 nM, 100 nM, or 10 nM.

In some embodiments, the HC and LC variable domain sequences are components of the same polypeptide chain.

In other embodiments, the HC and LC variable domain sequences are components of different polypeptide chains. For example, the plasma kallikrein binding protein is IgG, such as IgG1, IgG2, IgG3 or IgG4. A plasma kallikrein binding protein can be a soluble Fab (sFab).

In other implementations, the plasma kallikrein binding protein is Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab Includes fusions or other molecules that contain the antigen binding site of one of the binding proteins herein. The VH and VL regions of these Fabs are IgG, Fab, Fab2, Fab2′, scFv, PEGylated Fab, PEGylated scFv, PEGylated Fab2, VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC ::HSA+VH::CH1, HSA::LC+VH::CH1, or other suitable structures.

In one embodiment, the plasma kallikrein binding protein is a human or humanized antibody, or non-immunogenic in humans. For example, the protein includes one or more human antibody framework regions, eg, all human framework regions.

In one embodiment, the plasma kallikrein binding protein comprises a human Fc domain or an Fc domain that is at least 95%, 96%, 97%, 98% or 99% identical to a human Fc domain.

In one embodiment, the plasma kallikrein binding protein is a primate or primatizing antibody or is non-immunogenic in humans. For example, the protein includes one or more of the primate antibody framework regions, eg, all primate framework regions.

In one embodiment, the plasma kallikrein binding protein comprises a primate Fc domain or an Fc domain that is at least 95%, 96%, 97%, 98% or 99% identical to a primate Fc domain. "Primates" include humans (Homo sapiens), chimpanzees (Pan troglodytes and Pan paniscus (bonobos)), gorillas (Gorilla gorilla), gibbons, monkeys, lemurs, aye-ayes (Daubentonia madagascariensis) and tarsiers.

In one embodiment, the plasma kallikrein binding protein comprises a human framework region or a framework region that is at least 95%, 96%, 97%, 98% or 99% identical to a human framework region.

In some embodiments, the plasma kallikrein binding protein does not contain any sequences from mouse or rabbit (eg, not mouse or rabbit antibodies).

In certain embodiments, the binding protein (eg, an antibody such as a human antibody) comprises a heavy chain immunoglobulin variable domain sequence and a light chain immunoglobulin variable domain sequence, wherein the heavy chain immunoglobulin variable domain sequence is defined herein comprising one, two or three (e.g., three) CDR regions from a heavy chain variable domain of a protein described herein and/or a light chain immunoglobulin variable domain sequence described herein comprising 1, 2 or 3 (eg 3) CDR regions from the light chain variable domain of a protein in which the protein binds (eg binds and inhibits) plasma kallikrein.

In some embodiments, the heavy chain immunoglobulin variable domain sequence is M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to herein as DX-2922), X81-B01, X67 -D03, X67-G04, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X124-G01 (also referred to herein as DX-2930), one, two or three (eg, three) from the heavy chain variable domains of X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04 ) and/or the light chain immunoglobulin variable domain sequence is M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to herein as DX-2922), X81-B01, X67-D03, X67-G04, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115- 1, 2 or 3 from the light chain variable domain of F02, X124-G01 (also referred to herein as DX-2930), X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04 (each) including three (eg, three) CDR regions.

In certain embodiments, 1, 2 or 3 (eg 3) CDR regions from the heavy chain variable domain are from X81-B01 and/or 1, 2 from the light chain variable region One or three (eg, three) CDR regions are from X81-B01 or from X67-D03.

In certain embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of a protein described herein and/or the light chain immunoglobulin variable domain sequence comprises a protein described herein contains the light chain variable domain of

In some embodiments, the heavy chain immunoglobulin variable domain sequence is M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to herein as DX-2922), X81-B01, X67 -D03, X67-G04, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X124-G01 (also referred to herein as DX-2930), X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04 heavy chain variable domains and/or light chain immunoglobulin variable domain sequences M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to herein as DX-2922), X81-B01, X67-D03, X67-G04, X81-B01, X67 - D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X124-G01 (also referred to herein as DX-2930), including the light chain variable domains of X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04 (respectively).

In certain embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of X81-B01 and/or the light chain immunoglobulin variable domain sequence comprises a light chain variable domain of X81-B01.

In some embodiments, the heavy chain immunoglobulin variable domain sequence comprises a heavy chain variable domain of X67-D03, X101-A01, M162-A04, X115-F02, X124-G01 or X63-G06 and/or a light chain Immunoglobulin variable domain sequences include light chain variable domains of X67-D03, X101-A01, M162-A04, X115-F02, X124-G01 or X63-G06 (respectively).

In certain embodiments, the protein comprises a heavy chain of a protein described herein and/or a light chain of a protein described herein.

In some embodiments, the protein is M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to herein as DX-2922), X81-B01, X67-D03, X67- G04, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X124-G01 (herein DX-2930), containing heavy chains of X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04 (respectively).

In some embodiments, the protein comprises an X81-B01 heavy chain and/or an X81-B01 light chain.

In some embodiments, the protein is X67-D03, X101-A01, M162-A04, X115-F02, X124-G01 or X63-G06 heavy chain and/or X67-D03, X101-A01, M162-A04, including light chains of X115-F02, X124-G01 or X63-G06 (respectively).

In some embodiments, the protein comprises: (a) human CDRs or human framework regions; , one or more of the CDRs that are 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical ( (c) the LC immunoglobulin variable domain sequence is at least 85%, 88%, 89%, 90%, 91% with the CDRs of the LC variable domains described herein , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical CDRs comprising one or more (e.g., 1, 2 or 3) (d) the LC immunoglobulin variable domain sequence is at least 85%, 88%, 89%, 90%, 91%, an LC variable domain (e.g., entire or framework region or CDR) described herein; 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical; (e) the HC immunoglobulin variable domain sequence is an HC variable described herein; domains (e.g. whole or framework regions or CDRs) and at least 85%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, is 99% or 100% identical; (f) the protein binds to an epitope to which a protein described herein binds or competes for binding with a protein described herein; g) a primate CDR or primate framework region; (h) an HC immunoglobulin variable domain sequence that differs by at least one amino acid from CDR1 of an HC variable domain described herein, but by two or three (i) the HC immunoglobulin variable domain sequence differs from the CDR2 of the HC variable domains described herein by at least one amino acid, but by two, three (j) the HC immunoglobulin variable domain sequence is an HC variable domain sequence described herein comprising a CDR2 that differs by no more than 1, 4, 5, 6, 7 or 8 amino acids; differs from CDR3 by at least one amino acid contains a CDR3 that differs by no more than 2, 3, 4, 5 or 6 amino acids; (k) the LC immunoglobulin variable domain sequence is an LC variable domain sequence described herein; (l) an LC immunoglobulin variable domain sequence as described herein, comprising a CDR1 that differs from CDR1 by at least one amino acid, but by no more than 2, 3, 4, or 5 amino acids; (m) the LC immunoglobulin variable domain sequence comprises a CDR2 that differs from the CDR2 of the LC variable domain described herein by at least one amino acid, but by no more than two, three, or four amino acids; (n) an LC immunoglobulin variable comprising a CDR3 that differs by at least one amino acid from the CDR3 of the LC variable domain described in the literature, but by no more than 2, 3, 4, or 5 amino acids; the domain sequence differs by at least one amino acid from the LC variable domains (e.g., whole or framework regions or CDRs) described herein by 2, 3, 4, 5, 6, and (o) the HC immunoglobulin variable domain sequences differ from the HC variable domains (e.g., entire or framework regions) described herein. or CDRs) by at least one amino acid, but by no more than 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids including one or more of

In some embodiments, the protein has an apparent inhibition constant (K _i,app ) of less than 1000 nM, 500 nM, 100 nM or 10 nM.

In a preferred embodiment, the protein is M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to herein as DX-2922), X81-B01, X67-D03, X67- G04, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X124-G01 (herein DX-2930), X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04. .

In a preferred embodiment, the protein is M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to herein as DX-2922), X81-B01, X67-D03, X67- G04, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X124-G01 (herein DX-2930), X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to herein as DX-2922), X81-B01, X67-D03, X67- G04, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X124-G01 (herein DX-2930), X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.

In a preferred embodiment, the protein is M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to herein as DX-2922), X81-B01, X67-D03, X67- G04, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X124-G01 (herein DX-2930), an antibody having an antibody variable region of the light and/or heavy chain of an antibody selected from the group consisting of X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04 ( human antibodies).

In some embodiments, the plasma kallikrein binding protein does not bind prekallikrein (eg, human prekallikrein), but binds the active form of plasma kallikrein (eg, human plasma kallikrein).

In certain embodiments, the plasma kallikrein-binding protein compares factor XIIa and/or bradykinin production to standard, e.g., factor XIIa and/or bradykinin production under the same conditions in the absence of the protein. about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60% , about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or more than about 95%.

In certain embodiments, the plasma kallikrein-binding protein has an apparent inhibition constant (K _i,app ) of less than 1000 nM, 500 nM, 100 nM, or 10 nM.

In some embodiments, the HC and LC variable domain sequences are components of the same polypeptide chain.

The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, goals and advantages of the disclosure will be apparent from the description and drawings, and from the claims.

The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference.

FIG. 1 is a table showing the correlation between truncated HMWK and rheumatoid arthritis, Crohn's disease and ulcerative colitis.

The present disclosure is based on the unexpected discovery that elevated levels of truncated HMWK have been observed in patients with RA, CD or UC. In particular, high concentrations of truncated HMWK were observed in RA patients and moderate concentrations of truncated HMWK were observed in both CD and UC patients. Thus, to identify a subject having or at risk of developing an autoimmune disease, such as RA, UC or CD, based on the concentration of truncated HMWK in the subject's biological sample, autologous Disclosed herein are novel diagnostic and prognostic methods for monitoring immune disease progression and for determining efficacy of treatment for autoimmune disease in a subject. Also disclosed herein are methods for the treatment of such autoimmune diseases and other diseases associated with the plasma kallikrein (pKal) system using pKal inhibitors such as those described herein. be.

DEFINITIONS For convenience, certain terms used in the specification, examples and appended claims are defined here before further description of the present disclosure.

The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

The term "antibody" refers to a protein comprising at least one immunoglobulin variable domain or immunoglobulin variable domain sequence. For example, an antibody can comprise a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In other examples, the antibody comprises two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody" includes antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab') ₂ , Fd fragments, Fv fragments, scFv, and domain antibody (dAb) fragments (de Wildt et al. , Eur J Immunol. 1996, 26(3), 629-639)) as well as complete antibodies. Antibodies can have the structural characteristics of IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof). Antibodies can be from any source, but are preferably primate (human and non-human primates) and primatized.

The VH and VL regions can be further subdivided into regions of hypervariability, termed "complementarity determining regions ("CDR"), interspersed with regions that are more conserved, termed framework regions ("FR"). Framework regions and CDR ranges have been precisely defined (Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 and Chothia, C. et al. (1987) J. Mol. Biol. The Kabat definition is used herein. Each VH and VL typically consists of 3 CDRs and 4 FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 be done.

Each VH or VL chain of an antibody may further comprise all or part of a heavy or light chain constant region, thereby forming an immunoglobulin heavy chain or immunoglobulin light chain, respectively. In one embodiment, an antibody is a tetramer of two immunoglobulin heavy chains and two immunoglobulin light chains, wherein the immunoglobulin heavy chains and immunoglobulin light chains are interconnected, for example, by disulfide bonds. ing. In IgG, the heavy chain constant region comprises three immunoglobulin domains CH1, CH2 and CH3. The light chain constant region contains the CL domain. The heavy and light chain variable regions contain the binding domains that interact with antigen. The constant region of an antibody typically mediates binding of the antibody to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system. The light chains of immunoglobulins may be of the kappa or lambda type. In one embodiment, the antibody is glycosylated. Antibodies can be functional with respect to antibody dependent cytotoxicity and/or complement dependent cytotoxicity.

One or more regions of the antibody may be human or effectively human. For example, one or more of the variable regions may be human or effectively human. For example, one or more of the CDRs may be human, eg, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3. Each of the light chain CDRs may be human. The HC CDR3 may be human. One or more of the framework regions may be human, eg, HC or LC FR1, FR2, FR3 and FR4. For example, the Fc region can be human. In one embodiment, all of the framework regions are human, with the framework sequences of an antibody produced in, for example, a human somatic cell, such as an immunoglobulin-producing hematopoietic or non-hematopoietic cell. . In one embodiment, the human sequences are germline sequences, eg, encoded by a germline nucleic acid. In one embodiment, the selected Fab framework (FR) residues are converted to the amino acid type of the corresponding residue in the most similar primate germline gene, particularly the human germline gene. can do. One or more of the constant regions may be human or effectively human. For example, at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98% of an immunoglobulin variable domain, constant region, constant domain (CH1, CH2, CH3, CL1) or whole antibody 100% may be human or effectively human.

All or part of an antibody can be encoded by an immunoglobulin gene or segment thereof. Exemplary human immunoglobulin genes include the kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes and many immunoglobulin variable region genes. . Full-length immunoglobulin "light chains" (about 25 kDa or about 214 amino acids) are encoded by variable region genes (about 110 amino acids) at the NH2 terminus and either kappa or lambda constant region genes at the COOH terminus. A full-length immunoglobulin "heavy chain" (about 50 kDa or about 446 amino acids) similarly includes the variable region genes (about 116 amino acids) and other above-described genes such as γ (encoding about 330 amino acids). Encoded by one of the constant region genes. The length of human HC varies greatly as the HC CDR3 varies from approximately 3 amino acid residues to over 35 amino acid residues.

The term "antigen-binding fragment" of a full-length antibody refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to a target of interest. Examples of binding fragments encompassed by the term "antigen-binding fragment" of a full-length antibody are (i) Fab fragments, which are monovalent fragments consisting of the VL, VH, CL and CH1 domains; (ii) a disulfide at the hinge region; F(ab′) ₂ fragment, a bivalent fragment comprising two Fab fragments joined by a bridge; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) from the VL and VH domains of the single arm of the antibody; (v) a dAb fragment consisting of the VH domain (Ward et al. (1989) Nature 341:544-546), and (vi) an isolated complementarity determining region (CDR) that retains functionality. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they are joined by a synthetic linker using recombinant methods to pair the VL and VH regions. can be produced as a single protein chain forming a monovalent molecule known as a single-chain Fv (scFv) (e.g., US Pat. No. 5,260,203, US Pat. No. 4,946,778 and US Patent No. 4,881,175, Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).

Antibody fragments can be obtained using any suitable technique, including conventional techniques known to those of skill in the art. The term "monospecific antibody" refers to an antibody that displays a single binding specificity and binding affinity for a particular target, eg, epitope. This term includes a "monoclonal antibody" or "monoclonal antibody composition", which as used herein are preparations of antibodies or fragments thereof of single molecular composition, regardless of how the antibodies are produced. used to refer to

The inhibition constant (Ki) provides a measure of inhibitor potency, which is the concentration of inhibitor required to halve the enzyme activity, and is independent of enzyme or substrate concentration. Apparent Ki (K _i,app ) is obtained at different substrate concentrations by measuring the inhibitory effect of different concentrations of inhibitor (e.g. inhibitory binding protein) on the extent of the reaction (e.g. enzyme activity) to determine the effect of inhibition An estimate of the apparent Ki value is obtained by fitting the change in the pseudo-first order rate constant as a function of agent concentration to the Morrison equation (equation 1). Ki is obtained from the Y-intercept obtained from linear regression analysis of plots of K _i,app versus substrate concentration.

As used herein, "binding affinity" refers to the apparent association constant or _Ka . K _a is the reciprocal of the dissociation constant (K _d ). The binding protein is, for example, at least 10 ⁵ M ⁻¹ , 10 ⁶ M ⁻¹ , 10 ⁷ M ⁻¹ , 10 ⁸ M ⁻¹ , 10 ⁹ M ⁻¹ , 10 ¹⁰ M ⁻¹ to a particular target molecule. and a binding affinity of 10 ¹¹ M ⁻¹ . The higher affinity of binding protein binding to the first target relative to the second target indicates that the Ka for binding to the first target is _equal to the _Ka for binding to the second target (or the numerical value K _d ) (or a smaller numerical value K _d ). In such a case, the binding protein is a first target (e.g., a second proteins in one conformation (or their mimetics). A difference in binding affinity (e.g., specificity or other comparison) is at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 37.5-fold, 50 It can be 1-fold, 70-fold, 80-fold, 91-fold, 100-fold, ⁵⁰⁰ -fold, 1000-fold, or 105-fold.

Binding affinity can be measured by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance or spectroscopy (eg, using a fluorescence assay). Exemplary conditions for evaluating binding affinity are in TRIS buffer (50 mM TRIS, 150 mM NaCl, ₅ mM CaCl2, pH 7.5). These techniques can be used to measure the concentration of bound and free binding protein as a function of binding protein (or target) concentration. The concentration of bound binding protein ([Bound]) is the concentration of free bound protein ([Free]) and the concentration of binding sites for the binding protein on the target, combined with the following formula [Bound] = N [Free] / ((1/Ka) + [free])
where (N) is the number of binding sites per target molecule.

However, it is not always necessary to accurately determine the _Ka , because it is sometimes determined using methods such as ELISA or FACS analysis, and is proportional to the _Ka , thus higher affinities are , obtaining a quantitative measurement of affinity that can be used for comparison, e.g., determining whether it is two-fold higher, obtaining a qualitative measurement of However, it is sufficient to obtain an estimate of affinity by activity in, for example, a functional assay.

The term "binding protein" refers to a protein capable of interacting with a target molecule. This term is used interchangeably with "ligand". A "plasma kallikrein-binding protein" refers to a protein capable of interacting with (eg, binding to) plasma kallikrein, and particularly includes proteins that selectively or specifically interact with and/or inhibit plasma kallikrein. A protein inhibits plasma kallikrein if it causes a reduction in the activity of plasma kallikrein compared to the activity of plasma kallikrein under the same conditions in the absence of the protein. In some embodiments, the plasma kallikrein binding protein is an antibody.

The term "kallikrein inhibitor" refers to any agent or molecule that inhibits kallikrein.

The term "combination" refers to the use of two or more agents or treatments to treat the same patient where the times of use or action of the agents or treatments overlap. The agents or treatments can be administered simultaneously (eg, as one formulation administered to the patient or as two separate formulations administered simultaneously) or sequentially in any order.

A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine). , non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g. threonine, valine, isoleucine), as well as aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine).

One or more of the framework and/or CDR amino acid residues (or binding loop amino acid residues) of the binding protein have one or more mutations (e.g., substitutions) relative to the binding proteins described herein. (eg, conservative or non-essential amino acid substitutions), insertions or deletions). The plasma kallikrein binding protein has mutations (e.g., substitutions (e.g., conservative substitutions or non-essential amino acid substitutions) (e.g., at least 1, 2, 3 or 4) relative to the binding proteins described herein and/or or 15, 12, 10, 9, 8, 7, 6, 5, 4, 3 or less than 2), e.g. The mutation may be in the framework region, the CDR (or binding loop) and/or the constant region.In one embodiment, the mutation is in the framework region. In an embodiment, the mutation is in the CDRs.In some embodiments, the mutation is in the constant region.Whether or not a particular substitution is tolerated, i.e. adversely affects a biological property such as binding activity. Failure to give a can be predicted, for example, by assessing whether the mutation is conservative or by the method of Bowie et al. (1990) Science 247, 1306-1310.

A "virtually human" immunoglobulin variable region is an immunoglobulin variable region that contains a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in a normal human. A "virtually human" antibody is an antibody that contains a sufficient number of human amino acid positions such that the antibody does not elicit an immunogenic response in normal humans.

"Epitope" refers to the site on a target compound that is bound by a binding protein (eg, an antibody such as a Fab or full-length antibody). Where the target compound is a protein, the moieties may consist entirely of amino acid building blocks, or may consist entirely of chemical modifications of amino acids of the protein (e.g., glycosyl moieties), or consist of combinations thereof. can be done. Overlapping epitopes contain at least one amino acid residue, glycosyl group, phosphate group, sulfate group, or other molecular feature in common.

If the first binding protein binds at the same site on the target compound that the second binding protein binds, or overlaps with the site that the second binding protein binds (e.g., amino acid sequence or other molecular feature (e.g., amino acid sequence or other molecular feature) (e.g., glycosyl, phosphate or sulfate groups), e.g. A (eg, an antibody) "binds to the same epitope" as a second binding protein (eg, an antibody).

Binding of a first binding protein to its epitope reduces the amount of a second binding protein that binds to its epitope (e.g. 10%, 20%, 30%, 40%, 50%, 60%, 70%). %, 80%, 90%, 100% or more), the first binding protein (eg, antibody) "competes for binding" with the second binding protein (eg, antibody). Competition is direct (e.g., the first binding protein binds to the same epitope as the second binding protein binds, or the first binding protein overlaps the second binding protein's binding epitope). binding), even indirectly (e.g., binding to an epitope of a first binding protein causes a conformational change in the target compound, thereby reducing the ability of a second binding protein to bind its epitope) may be

Calculations of "homology" or "sequence identity" between two sequences (the terms are used interchangeably herein) are performed as follows. The sequences are aligned for the purpose of optimal comparison (e.g., for optimal alignment, gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences can be used for comparison). can be disregarded for purposes). The best alignment is determined as the best score using the GAP program in the GCG software package according to the Blossom62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein sometimes amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between two sequences is a function of the number of identical positions shared by the sequences.

In preferred embodiments, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even More preferably at least 70%, 80%, 90%, 92%, 95%, 97%, 98% or 100%. For example, the reference sequence can be the length of an immunoglobulin variable domain sequence.

A "humanized" immunoglobulin variable region is an immunoglobulin variable region that has been altered to contain a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not elicit an immunogenic response in normal humans. . Descriptions of "humanized" immunoglobulins include, for example, US Pat. No. 6,407,213 and US Pat. No. 5,693,762.

As used herein, the term "hybridizing under low, moderate, high or very high stringency conditions" describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in "Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6." Aqueous and non-aqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: (1) in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by at least 50° C. (under low stringency conditions); (2) low stringency hybridization conditions with two washes in 0.2X SSC, 0.1% SDS at approximately 45°C; medium stringent hybridization conditions in SSC followed by one or more washes in 0.2×SSC at 60° C., 0.1% SDS; (3) 6×SSC at about 45° C. (4) very high stringency hybridization conditions followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.; perform one or more washes in 0.5 M sodium phosphate, 7% SDS at 65°C, followed by one or more washes in 0.2 x SSC, 1% SDS at 65°C. Very high stringency conditions (4) are the preferred conditions and should be used unless otherwise specified. The present disclosure hybridizes at low, medium, high or very high stringency to a nucleic acid described herein or a complement thereof, such as a nucleic acid encoding a binding protein described herein. contains nucleic acids that The nucleic acid may be the same length as the reference nucleic acid, or within 30%, 20% or 10% of that length. The nucleic acids may correspond to regions encoding the immunoglobulin variable domains described herein.

"Isolated composition" refers to a composition from which at least 90% of at least one component of the natural sample from which the isolated composition was obtained has been removed. An artificially or naturally produced composition contains at least 5, 10, 25, 50, 75, 80%, 90%, 92%, 95%, 98% or 99% of the species or population of species of interest. When weight percent pure, it may be a "composition" that is "at least" to a certain degree of purity.

An "isolated" protein refers to a protein that has been removed from at least 90% of at least one component of the natural sample from which the isolated protein was obtained. A protein is "at least" to a certain degree pure if the species or collection of species of interest is at least 5, 10, 25, 50, 75, 80, 90, 92, 95, 98 or 99% pure by weight. can be

A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of a binding agent, e.g., an antibody, without abolishing, or more preferably substantially altering, the biological activity, whereas Altering an "essential" amino acid residue in a results in a large loss of activity.

A "patient", "subject" or "host" (the terms are used interchangeably) to be treated by the method can mean either a human or non-human animal.

A "subject in need thereof" includes, for example, a subject having a disease or disorder described herein or a subject at risk of developing a disease or disorder described herein.

The term "kallikrein" (eg, plasma kallikrein) refers to peptidases (enzymes that cleave peptide bonds in proteins), a subgroup of the serine protease family. Plasma kallikrein cleaves kininogen to produce kinin, a potent pro-inflammatory peptide. DX-88 (also referred to herein as "PEP-1") is a potent (Ki<1 nM) and specific inhibitor of plasma kallikrein (NP_000883) (see, eg, WO95/21601 or WO2003/103475). .

The amino acid sequence of KLKb1 (plasma kallikrein) is
KLKb1
>gi|78191798|ref|NP_000883.2| Plasma kallikrein B1 precursor [Homo sapiens]
ＭＩＬＦＫＱＡＴＹＦＩＳＬＦＡＴＶＳＣＧＣＬＴＱＬＹＥＮＡＦＦＲＧＧＤＶＡＳＭＹＴＰＮＡＱＹＣＱＭＲＣＴＦＨＰＲＣＬＬＦＳＦＬＰＡＳＳＩＮＤＭＥＫＲＦＧＣＦＬＫＤＳＶＴＧＴＬＰＫＶＨＲＴＧＡＶＳＧＨＳＬＫＱＣＧＨＱＩＳＡＣＨＲＤＩＹＫＧＶＤＭＲＧＶＮＦＮＶＳＫＶＳＳＶＥＥＣＱＫＲＣＴＳＮＩＲＣＱＦＦＳＹＡＴＱＴＦＨＫＡＥＹＲＮＮＣＬＬＫＹＳＰＧＧＴＰＴＡＩＫＶＬＳＮＶＥＳＧＦＳＬＫＰＣＡＬＳＥＩＧＣＨＭＮＩＦＱＨＬＡＦＳＤＶＤＶＡＲＶＬＴＰＤＡＦＶＣＲＴＩＣＴＹＨＰＮＣＬＦＦＴＦＹＴＮＶＷＫＩＥＳＱＲＮＶＣＬＬＫＴＳＥＳＧＴＰＳＳＳＴＰＱＥＮＴＩＳＧＹＳＬＬＴＣＫＲＴＬＰＥＰＣＨＳＫＩＹＰＧＶＤＦＧＧＥＥＬＮＶＴＦＶＫＧＶＮＶＣＱＥＴＣＴＫＭＩＲＣＱＦＦＴＹＳＬＬＰＥＤＣＫＥＥＫＣＫＣＦＬＲＬＳＭＤＧＳＰＴＲＩＡＹＧＴＱＧＳＳＧＹＳＬＲＬＣＮＴＧＤＮＳＶＣＴＴＫＴＳＴＲＩＶＧＧＴＮＳＳＷＧＥＷＰＷＱＶＳＬＱＶＫＬＴＡＱＲＨＬＣＧＧＳＬＩＧＨＱＷＶＬＴＡＡＨＣＦＤＧＬＰＬＱＤＶＷＲＩＹＳＧＩＬＮＬＳＤＩＴＫＤＴＰＦＳＱＩＫＥＩＩＩＨＱＮＹＫＶＳＥＧＮＨＤＩＡＬＩＫＬＱＡＰＬＮＹＴＥＦＱＫＰＩＣＬＰＳＫＧＤＴＳＴＩＹＴＮＣＷＶＴＧＷＧＦＳＫＥＫＧＥＩＱＮＩＬＱＫＶＮＩＰＬＶＴＮＥＥＣＱＫＲＹＱＤＹＫＩＴＱＲＭＶＣＡＧＹＫＥＧＧＫＤＡＣＫＧＤＳＧＧＰＬＶＣＫＨＮＧＭＷＲＬＶＧＩＴＳＷＧＥＧＣＡＲＲＥＱＰＧＶＹＴＫＶＡＥＹＭＤＷＩＬＥＫＴＱＳＳＤＧＫＡＱＭＱＳＰＡ（配列識別番号３）である。

As used herein, the term "DX-2922" is used interchangeably with the term "X101-A01." Other variants of this antibody are described below.

As used herein, the term "DX-2930" is used interchangeably with the term "X124-G01." Other variants of this antibody are described below.

KLK1
>gi|13529059|gb|AAH05313.1|Kallikrein 1 [Homo sapiens]
ＭＷＦＬＶＬＣＬＡＬＳＬＧＧＴＧＡＡＰＰＩＱＳＲＩＶＧＧＷＥＣＥＱＨＳＱＰＷＱＡＡＬＹＨＦＳＴＦＱＣＧＧＩＬＶＨＲＱＷＶＬＴＡＡＨＣＩＳＤＮＹＱＬＷＬＧＲＨＮＬＦＤＤＥＮＴＡＱＦＶＨＶＳＥＳＦＰＨＰＧＦＮＭＳＬＬＥＮＨＴＲＱＡＤＥＤＹＳＨＤＬＭＬＬＲＬＴＥＰＡＤＴＩＴＤＡＶＫＶＶＥＬＰＴＱＥＰＥＶＧＳＴＣＬＡＳＧＷＧＳＩＥＰＥＮＦＳＦＰＤＤＬＱＣＶＤＬＫＩＬＰＮＤＥＣＫＫＶＨＶＱＫＶＴＤＦＭＬＣＶＧＨＬＥＧＧＫＤＴＣＶＧＤＳＧＧＰＬＭＣＤＧＶＬＱＧＶＴＳＷＧＹＶＰＣＧＴＰＮＫＰＳＶＡＶＲＶＬＳＹＶＫＷＩＥＤＴＩＡＥＮＳ（配列識別番号４）

As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, and , intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, dural Includes, but is not limited to, extramembranous and intrasternal injections and infusions.

The term "preventing" a disease in a subject refers to subjecting the subject to drug treatment, e.g., administration of a drug, such that at least one symptom of the disease is prevented, i. It is administered prior to clinical manifestation of the undesirable condition (eg, disease or other undesirable condition of the host animal) to prevent the condition from occurring. "Preventing" a disease is also called "prevention" or "prophylactic treatment."

A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Because prophylactic doses are typically used in subjects prior to disease or in the early stages of disease, the prophylactically effective amount may, but need not be, less than the therapeutically effective amount. .

As used herein, the term "substantially identical" (or "substantially homologous") means that the first and second amino acid or nucleic acid sequences exhibit similar activity, e.g., binding activity, binding selectivity, Alternatively, a first amino acid or nucleic acid sequence has a sufficient number of identical or equivalent (e.g., conservative amino acids) to a second amino acid or nucleic acid sequence such that it has (or encodes a protein with) biological activity. It is used herein to refer to including amino acid residues or nucleic acids (with similar side chains such as substitutions). In the case of antibodies, the second antibody has the same specificity for the same antigen and has at least 50%, at least 25% or at least 10% affinity for the same antigen.

Sequences similar or homologous (eg, having at least about 85% sequence identity) to the sequences disclosed herein are also part of this application. In some embodiments, the sequence identity is about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more good. In some embodiments, the plasma kallikrein-binding protein is about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the present It may have sequence identity to the binding proteins described herein. In certain embodiments, the plasma kallikrein binding protein has a They may have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity. In certain embodiments, the plasma kallikrein binding protein is about 85% in HC and/or LC CDRs (e.g., HC and/or LC CDR1, 2 and/or 3) relative to the binding proteins described herein, They may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity. In certain embodiments, the plasma kallikrein binding protein has about 85%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.

Moreover, substantial identity exists when the nucleic acid segments bind to complementary strands under selective hybridization conditions (eg, highly stringent hybridization conditions). Nucleic acids can be present in whole cells, in cell lysates, or in partially purified or substantially pure form.

A biopolymer motif sequence can include positions that can be mutated amino acids. For example, the symbol "X" in such contexts refers to any amino acid (eg, any of the twenty naturally occurring amino acids) unless otherwise specified, eg, any non-cysteine amino acid. Other permissible amino acids can also be designated using, for example, brackets and slashes. For example, "A/W/F/N/Q" means that alanine, tryptophan, phenylalanine, asparagine and glutamine are allowed at that particular position.

Statistical significance can be determined by any art-known method. Exemplary statistical tests include the Student's t-test, the Mann-Whitney U nonparametric test, and the Wilcoxon nonparametric test. A statistically significant relationship has a P-value less than 0.05 or 0.02. A particular binding protein may exhibit a statistically significant (eg, P value <0.05 or 0.02) difference, eg, in specificity or binding. The terms "induce", "inhibit", "enhance", "raise", "increase", "decrease", etc. refer to a distinguishable qualitative or quantitative state, e.g., between two states. Denotes a difference and may refer to a difference between two states, eg, a statistically significant difference.

A "therapeutically effective amount" refers to an amount, at dosages and for periods of time necessary, sufficient to achieve the desired therapeutic result. A therapeutically effective amount of a composition may vary according to factors such as the individual's disease state, age, sex and weight, and the ability of the protein to elicit the desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects.

A "therapeutically effective dose" preferably modulates a measurable parameter of the disease or disorder. For example, a therapeutically effective dose reduces the degree of symptoms of a disease or disorder by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and Even more preferably, it can be reduced by about 80%. The ability of a compound to modulate a measurable parameter, such as a parameter associated with disease, can be evaluated in animal model systems predictive of efficacy in human disorders and conditions. Alternatively, this property of a composition can be evaluated by testing the compound's ability to modulate the parameter in vitro.

"Treatment" of a disease or disorder in a subject (including, e.g., "treatment" of a subject who has the disease or disorder or is at risk of developing the disease or disorder) includes administering drug therapy, e.g., administering a drug, to the subject. means prevention, cure, alleviation, reduction, etc., of at least one symptom of a disease.

A "disorder associated with protein misfolding or aggregation" is a disease that results, at least in part, from changes in the protein folding process or changes in the stability of the protein fold. Impairments in the folding process, increased aggregation, and destabilization of the native protein structure can lead to loss-of-function or gain-of-function pathologies. Diseases associated with protein misfolding or aggregation include, but are not limited to, systemic amyloidosis, cryoglobulinemia, and sickle cell disease. Neurological diseases associated with protein misfolding or aggregation include other amyloid diseases such as Jakob-Creutzfeldt disease (associated with prion protein), Alzheimer's disease, familial amyloid polyneuropathy (FAP).

I. Use of truncated high molecular weight kininogen (HMWK) as a biomarker in diagnostic and prognostic assays for autoimmune diseases Elevated concentrations of truncated HMWK have been found in autoimmune diseases such as. See Example 1 below. Thus, truncated HMWKs serve as reliable biomarkers for diagnosing autoimmune diseases (e.g., RA, UC and CD), monitoring the progression of such autoimmune diseases, and determining therapeutic efficacy of diseases. can be fulfilled.

Accordingly, methods for the diagnosis and prognosis of autoimmune diseases (e.g., RA, UC and CD) based on the concentration of truncated HMWK in a biological sample (e.g., plasma sample) obtained from a candidate patient are provided herein. be written.

High molecular weight kininogen (HMWK), also known as Williams-Fitzgerald-Flaujeac factor, Fitzgerald factor, HMWK-kallikrein factor, is a protein from the blood coagulation system as well as the kinin-kallikrein system. It is a protein that adsorbs in vivo to biomaterial surfaces in contact with blood. High molecular weight kininogen (HMWK) exists in plasma as a single polypeptide (single chain) multidomain (1-6 domains) protein with a molecular weight of approximately 110 kDa. HMWK is cleaved by pKal in domain 4, releasing the 9 amino acid pro-inflammatory peptide bradykinin and a two-chain form of HMWK (truncated kininogen). The two chains of HMWK are the heavy chain containing domains 1-3 of HMWK and the light chain with domains 5 and 6 of HMWK. Heavy and light chains have molecular weights of approximately 56 and 46 kilodaltons, respectively.

The human gene encoding HMWK is Kininogen 1 (KNG1). KNG1 is transcribed and alternatively spliced to form mRNAs encoding either HMWKs or low molecular weight kininogens (LMWKs). An exemplary protein sequence for HMWK is provided below:
>gi|156231037|ref|NP_001095886.1|Kininogen-1 isoform 1 precursor [Homo sapiens]
ＭＫＬＩＴＩＬＦＬＣＳＲＬＬＬＳＬＴＱＥＳＱＳＥＥＩＤＣＮＤＫＤＬＦＫＡＶＤＡＡＬＫＫＹＮＳＱＮＱＳＮＮＱＦＶＬＹＲＩＴＥＡＴＫＴＶＧＳＤＴＦＹＳＦＫＹＥＩＫＥＧＤＣＰＶＱＳＧＫＴＷＱＤＣＥＹＫＤＡＡＫＡＡＴＧＥＣＴＡＴＶＧＫＲＳＳＴＫＦＳＶＡＴＱＴＣＱＩＴＰＡＥＧＰＶＶＴＡＱＹＤＣＬＧＣＶＨＰＩＳＴＱＳＰＤＬＥＰＩＬＲＨＧＩＱＹＦＮＮＮＴＱＨＳＳＬＦＭＬＮＥＶＫＲＡＱＲＱＶＶＡＧＬＮＦＲＩＴＹＳＩＶＱＴＮＣＳＫＥＮＦＬＦＬＴＰＤＣＫＳＬＷＮＧＤＴＧＥＣＴＤＮＡＹＩＤＩＱＬＲＩＡＳＦＳＱＮＣＤＩＹＰＧＫＤＦＶＱＰＰＴＫＩＣＶＧＣＰＲＤＩＰＴＮＳＰＥＬＥＥＴＬＴＨＴＩＴＫＬＮＡＥＮＮＡＴＦＹＦＫＩＤＮＶＫＫＡＲＶＱＶＶＡＧＫＫＹＦＩＤＦＶＡＲＥＴＴＣＳＫＥＳＮＥＥＬＴＥＳＣＥＴＫＫＬＧＱＳＬＤＣＮＡＥＶＹＶＶＰＷＥＫＫＩＹＰＴＶＮＣＱＰＬＧＭＩＳＬＭＫＲＰＰＧＦＳＰＦＲＳＳＲＩＧＥＩＫＥＥＴＴＶＳＰＰＨＴＳＭＡＰＡＱＤＥＥＲＤＳＧＫＥＱＧＨＴＲＲＨＤＷＧＨＥＫＱＲＫＨＮＬＧＨＧＨＫＨＥＲＤＱＧＨＧＨＱＲＧＨＧＬＧＨＧＨＥＱＱＨＧＬＧＨＧＨＫＦＫＬＤＤＤＬＥＨＱＧＧＨＶＬＤＨＧＨＫＨＫＨＧＨＧＨＧＫＨＫＮＫＧＫＫＮＧＫＨＮＧＷＫＴＥＨＬＡＳＳＳＥＤＳＴＴＰＳＡＱＴＱＥＫＴＥＧＰＴＰＩＰＳＬＡＫＰＧＶＴＶＴＦＳＤＦＱＤＳＤＬＩＡＴＭＭＰＰＩＳＰＡＰＩＱＳＤＤＤＷＩＰＤＩＱＩＤＰＮＧＬＳＦＮＰＩＳＤＦＰＤＴＴＳＰＫＣＰＧＲＰＷＫＳＶＳＥＩＮＰＴＴＱＭＫＥＳＹＹＦＤＬＴＤＧＬＳ（配列識別番号５）

Intact high molecular weight kininogen (HMWK) can be assayed using, for example, agglutination methods or immunological methods, such as radioimmunoassays (eg Kerbiriou-Nabias, DM, Br J Haematol, 1984, 56 (2 ): 2734-2786). Monoclonal antibodies against the light chain of human HMWK are known (see, eg, Reddigari, SR and Kaplan, AP, Blood, 1999, 74:695-702). Assays for HMWK based on chromogenic substrates can also be used (eg Scott, CF et al. Thromb Res. 1987, 48(6):685-700; Gallimore, M. J. et al. Thromb Res. 2004, 114). (2):91-96).

Truncated high molecular weight kininogen (HMWK), also referred to herein as "truncated kininogen", can be assayed, for example, using the methods described in Example 1, such as, for example, Western blot. Antibodies that specifically bind to truncated HMWK can be used, eg, mouse mAb clone 11H05. Additionally, truncated HMWKs may be evaluated using mass spectrometry. Immunoblot techniques for assessing the concentration of truncated HMWK are known in the art (see, eg, Buhler R. et al., Blood Coagul Fibrinolysis, 1995, 6(3):223-232).

Exemplary sequences for heavy and light chains of truncated kininogen are provided below.
＞ 切断型キニノーゲン－１重鎖ＱＥＳＱＳＥＥＩＤＣＮＤＫＤＬＦＫＡＶＤＡＡＬＫＫＹＮＳＱＮＱＳＮＮＱＦＶＬＹＲＩＴＥＡＴＫＴＶＧＳＤＴＦＹＳＦＫＹＥＩＫＥＧＤＣＰＶＱＳＧＫＴＷＱＤＣＥＹＫＤＡＡＫＡＡＴＧＥＣＴＡＴＶＧＫＲＳＳＴＫＦＳＶＡＴＱＴＣＱＩＴＰＡＥＧＰＶＶＴＡＱＹＤＣＬＧＣＶＨＰＩＳＴＱＳＰＤＬＥＰＩＬＲＨＧＩＱＹＦＮＮＮＴＱＨＳＳＬＦＭＬＮＥＶＫＲＡＱＲＱＶＶＡＧＬＮＦＲＩＴＹＳＩＶＱＴＮＣＳＫＥＮＦＬＦＬＴＰＤＣＫＳＬＷＮＧＤＴＧＥＣＴＤＮＡＹＩＤＩＱＬＲＩＡＳＦＳＱＮＣＤＩＹＰＧＫＤＦＶＱＰＰＴＫＩＣＶＧＣＰＲＤＩＰＴＮＳＰＥＬＥＥＴＬＴＨＴＩＴＫＬＮＡＥＮＮＡＴＦＹＦＫＩＤＮＶＫＫＡＲＶＱＶＶＡＧＫＫＹＦＩＤＦＶＡＲＥＴＴＣＳＫＥＳＮＥＥＬＴＥＳＣＥＴＫＫＬＧＱＳＬＤＣＮＡＥＶＹＶＶＰＷＥＫＫＩＹＰＴＶＮＣＱＰＬＧＭＩＳＬＭＫ （配列識別番号６）
＞ 切断型キニノーゲン－１軽鎖ＳＳＲＩＧＥＩＫＥＥＴＴＶＳＰＰＨＴＳＭＡＰＡＱＤＥＥＲＤＳＧＫＥＱＧＨＴＲＲＨＤＷＧＨＥＫＱＲＫＨＮＬＧＨＧＨＫＨＥＲＤＱＧＨＧＨＱＲＧＨＧＬＧＨＧＨＥＱＱＨＧＬＧＨＧＨＫＦＫＬＤＤＤＬＥＨＱＧＧＨＶＬＤＨＧＨＫＨＫＨＧＨＧＨＧＫＨＫＮＫＧＫＫＮＧＫＨＮＧＷＫＴＥＨＬＡＳＳＳＥＤＳＴＴＰＳＡＱＴＱＥＫＴＥＧＰＴＰＩＰＳＬＡＫＰＧＶＴＶＴＦＳＤＦＱＤＳＤＬＩＡＴＭＭＰＰＩＳＰＡＰＩＱＳＤＤＤＷＩＰＤＩＱＩＤＰＮＧＬＳＦＮＰＩＳＤＦＰＤＴＴＳＰＫＣＰＧＲＰＷＫＳＶＳＥＩＮＰＴＴＱＭＫＥＳＹＹＦＤＬＴＤＧＬＳ （配列識別番号７）

In certain instances, the concentrations of intact HMWK and truncated HMWK are measured by Western blot analysis, eg, Simple Western™ Protein Simple® Western blot analysis. Ｓｉｍｐｌｅ Ｗｅｓｔｅｒｎ（商標）アッセイは、当技術分野において公知である（例えば、Ｒｕｓｔａｎｄｉら、「Ｑｕａｌｉｔａｔｉｖｅ ａｎｄ ｑｕａｎｔｉｔａｔｉｖｅ ｅｖａｌｕａｔｉｏｎ ｏｆ Ｓｉｍｏｎ（商標）， ａ ｎｅｗ ＣＥ－ｂａｓｅｄ ａｕｔｏｍａｔｅｄ Ｗｅｓｔｅｒｎ ｂｌｏｔ ｓｙｓｔｅｍ ａｓ ａｐｐｌｉｅｄ ｔｏ ｖａｃｃｉｎｅ ｄｅｖｅｌｏｐｍｅｎｔ．」、Ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ 2012 Sep;33(17):2790-2797). Simple Western(TM) products are also commercially available (see, for example, ProteinSimple(R) of Santa Clara, Calif.).

A biological sample (e.g., plasma or serum sample) for measuring the concentration of truncated HMWK in order to perform any of the diagnostic and/or prognostic methods described herein body fluid) can be obtained from a candidate subject (eg, a candidate human patient). The subject may be a mammal, more preferably a human. Non-human mammals include, but are not limited to farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. A human subject may be a human patient suspected of having an autoimmune disease, such as those described herein such as RA, CD or UC.

A biological sample obtained from a subject may be a tissue or fluid sample. Examples of fluid samples include, but are not limited to saliva, blood, plasma, serum and urine. In certain embodiments, the biological sample from the subject contains white blood cells, eg, a blood sample. A biological sample can be obtained using any method known in the art, such as, for example, venipuncture, biopsy or swab. A protease inhibitor or protease inhibitor cocktail to inhibit HMWK cleavage in vitro can be added to the biological sample prior to analysis. Any protease inhibitor known in the art can be used in the methods described herein.

The concentration of truncated HMWK can be measured by any suitable assay known in the art (e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001, Current Protocols in Molecular Biology, FM Ausubel et al., eds., John Wiley & Sons, Inc., New York; Protocols in Molecular Biology, edited by FM Ausubel et al., John Wiley & Sons, Inc., New York).

In some embodiments, the concentration of truncated HMWK protein in the sample is measured. Assays to measure truncated HMWK protein concentration include immunoassays (e.g., herein referred to as immuno-based or immuno-based assays such as Western blots, immunohistochemistry and ELISA assays), mass spectrometry, and multiplex beads. Including but not limited to base assays. Assays for such protein concentration detection are known in the art.

In one example, the concentration of truncated HMWK is measured by a Western blot assay, which can include LiCor detection, as described herein. In other examples, the concentration of truncated HMWK protein can include binding partners such as antibodies that specifically bind truncated HMWK or specifically bind truncated HMWK and uncleaved HMWK. Measured by histochemical assay.

Binding partners for protein detection can be designed using methods known in the art and methods described herein. In some embodiments, a truncated HMWK protein binding partner, such as an anti-truncated HMWK antibody, binds to part or all of the amino acid sequence of the HMWK protein. Other examples of protein detection and quantification methods are described, e.g., in US Pat. Nos. 6,939,720 and 8,148,171, and multiplex Immunoassays, as well as protein microarrays, eg, as described in US Patent Application Publication No. 2009/0088329. Any suitable binding partner for truncated HMWK is contemplated for detecting truncated HMWK levels. In some embodiments, a binding partner is any molecule that specifically binds to a HMWK protein or truncated HMWK protein. Such binding partners can bind to truncated forms of HMWK (eg, truncated HMWK) with higher affinity than their binding to uncleaved HMWK. In some instances, a binding partner, such as an antibody, can only bind to truncated forms of HMWK.

Antibodies to be used in the methods described herein can be full length antibodies or Fab, F(ab)2, Fv, single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, It may be in any form, including but not limited to antigen binding fragments thereof, such as scFv or dAb fragments. Methods of making antibodies are well known in the art (see, e.g., Sambrook et al., "Molecular Cloning: A Laboratory Manual" (2nd ed.), Cold Spring Harbor Laboratory Press (1989), Lewin "Genes IV", Oxford University Press). , New York (1990) and Roitt et al., Immunology (2nd ed.), Gower Medical Publishing, London, New York (1989), WO2006/040153, WO2006/122786 and WO2003/002609. available for reference).

In other embodiments, the binding partner used to measure the concentration of truncated HMWK may be a non-antibody peptide molecule or aptamer that specifically binds to truncated HMWK. Methods of making peptide molecules and aptamers are also known in the art (see, for example, US Patent Application Publication No. 2009/0075834, US Patent No. 7435542, US Patent No. 7807351 and US Patent No. 7239742). ).

Once the concentration of truncated HMWK in a biological sample obtained from a candidate subject is determined, the subject has, is at risk for, or has an autoimmune disease, such as RA, CD or UC. It can be compared to control concentrations to determine if one is suspected of having them.

In certain embodiments, the control concentration is the amount of truncated HMWK in a control sample such as a cell, tissue or fluid obtained from a healthy subject or population of healthy subjects, preferably of the same species as the candidate subject. concentration. As used herein, a healthy subject is a subject who apparently does not have the target disease (e.g., RA, CD or UC) or has no history of the disease when the concentration of HMWK is measured. be.

In some embodiments, the control concentration is either undetectable using standard detection methods (e.g., Western blot or immunohistochemistry), or is below the background/noise level obtained using standard detection methods. Lower concentration of truncated HMWK. Preferably, the standard detection method is the same method used to measure the concentration of truncated HMWK in the sample of the candidate subject.

The control concentration may be the predetermined concentration. Such predetermined concentrations may represent concentrations of truncated HMWK in a population of subjects who do not have or are not at risk for an autoimmune disease as described herein. The predetermined concentration can take various forms. For example, it can be a single cutoff value such as the median or mean. In certain embodiments, such predetermined concentrations are one defined group known to have the target autoimmune disease (e.g., RA, UC or CD) and those without the target autoimmune disease. can be set based on a comparison group such as any other defined group for which is known. Alternatively, the predetermined concentration may be a range, eg, a range representing concentrations of truncated HMWK in a control population within a predetermined percentile.

Predetermined concentrations may depend on the particular population selected. For example, an apparently healthy person (with no detectable disease or history of a target autoimmune disease such as RA, CD or UC) should have a member with the target autoimmune disease. or have a different "normal" range of truncated HMWK than populations that are at risk of or may be in remission. The default concentration selected may therefore take into account the category the subject falls into. Appropriate ranges and categories can be selected by those skilled in the art with no more than routine experimentation.

Concentrations of the controls described herein can be determined by routine techniques. In some instances, the concentration of the control is determined by a conventional method for the control sample as described herein (e.g., by obtaining the concentration of truncated HMWK in the test sample as described herein). same assay). In another example, the concentration of truncated HMWK can be obtained from members of a control population, and the results analyzed, e.g., by a computer program, to represent the concentration of truncated HMWK in the control population ( given concentration) can be obtained.

The candidate subject has or is at risk of having the target autoimmune disease by comparing the concentration of truncated HMWK in a sample obtained from the candidate subject to the concentration of a control described herein. can decide whether For example, if the candidate subject's concentration of truncated HMWK deviates from the control concentration (e.g., increases or decreases relative to the control concentration), the candidate subject has or is at risk for the target autoimmune disease. can be identified as having

As used herein, "elevated concentration or concentration above control" means that the concentration of truncated HMWK is higher than the concentration of a control, such as a predetermined threshold or concentration of truncated HMWK in a control sample. means Control concentrations are described in more detail herein. Elevated concentrations of truncated HMWK are, e.g. , including concentrations of truncated HMWK greater than 150%, 200%, 300%, 400%, 500% or more. Elevated concentrations of truncated HMWK may also increase from a zero state (e.g., no or no truncated HMWK in the control) to a non-zero state (e.g., some truncated HMWK in the sample or with detectable truncated HMWK).

As used herein, a "reduced concentration or concentration below a control" means that the concentration of truncated HMWK is below a predetermined threshold or concentration of a control, such as the concentration of truncated HMWK in a control sample. means Control concentrations are described in more detail herein. Decreased concentrations of truncated HMWK, e.g. , including concentrations of truncated HMWK below 150%, 200%, 300%, 400%, 500% or more. Decreased concentrations of truncated HMWK may also indicate that a phenomenon goes from a non-zero state (e.g., some truncated HMWK or detectable truncated HMWK in the sample) to a zero state (e.g., control truncated HMWK is absent or undetectable).

In certain embodiments, a high concentration (e.g., at least 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of truncated HMWK is present in a biological sample obtained from a patient candidate for RA. If observed, the candidate is diagnosed as having or at risk for RA inflammation. If a moderate concentration (eg, about 10-30%) of truncated HMWK is observed in a biological sample obtained from a patient with a UC or CD candidate, the candidate has or is at risk of having UC or CD. diagnosed as having sex.

Additionally, the concentration of truncated HMWK can be used as a biomarker to monitor the progression of autoimmune diseases such as RA, UC and CD. For example, at least two biological samples (e.g., serum or plasma samples) at different time points from a human subject having or at risk of developing a target autoimmune disease such as RA, UC or CD. can be obtained. In some examples, the second biological sample can be obtained at least one month (eg, 3 months, 6 months, 9 months, or 12 months) after the first biological sample is obtained. The concentration of truncated HMWK can be measured in at least two biological samples. If the concentration of truncated HMWK increases over time (e.g., the concentration of truncated HMWK in a later obtained biological sample is at least 20%, 50% greater than the concentration in a previously obtained biological sample). %, 70%, 90%, 1-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold higher), disease progression in the subject (e.g., the risk of developing an autoimmune disease in the subject or exacerbation of autoimmune disease).

Moreover, the concentration of truncated HMWKs can be used as a biomarker to determine a subject's responsiveness to treatment for autoimmune diseases, such as those described herein. For example, multiple biological samples can be obtained from a human patient undergoing treatment during the course of treatment, and the concentration of truncated HMWK can be measured according to routine techniques such as those described herein. If the concentration of truncated HMWK in a human patient undergoing treatment remains decreased over the treatment period (e.g., the concentration of truncated HMWK in a biological sample obtained later than that obtained earlier). (e.g., at least 20%, 50%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 50-fold or 100-fold lower than the concentration in the biological sample), human Indicates that the patient is responsive to treatment. On the other hand, if the concentration of truncated HMWK remains substantially the same over the treatment period (e.g., the concentration of truncated HMWK in a later obtained biological sample is or less than 20%, such as 15%, 10% or 5% less than the concentration in the medium), indicates that the human patient is not responsive to treatment.

Suitable for treating disease when a subject is identified as having or at risk of having an autoimmune disease (e.g., RA, UC or CD) by any of the methods described herein treatment can be implemented. In certain instances, a subject can be treated with one or more pKal inhibitors described herein. When a subject is determined to be unresponsive to treatment by any method described herein, a higher dose and/or more frequent treatment (e.g., pKal inhibitor) can be administered to the subject. Alternatively, the subject can be switched to a different treatment. On the other hand, in subjects identified as responsive to treatment or in need of no further treatment, the dose or frequency of administration of the therapeutic agent is maintained, reduced or stopped.

II. Treatment of Autoimmune Diseases Also included herein are diabetic macular edema, retinal proliferation, brain trauma, acute spinal cord injury, focal amyloidosis, psoriasis, multiple sclerosis, inflammatory colitis, rheumatoid arthritis, vasculitis. associated with the plasma kallikrein (pKal) system, including but not limited to autoimmune diseases such as systemic lupus erythematosus nephritis, systemic mastocytosis, severe burns, and neuropathic pain (diabetic and postherpetic neuralgia) A method is described for treating a disease that causes Such methods include administering an effective amount of one or more kallikrein inhibitors to a subject in need of treatment (e.g., a human patient having or at risk for the disease) by a suitable route. including administering.

(A) Plasma Kallikrein Exemplary plasma kallikrein sequences against which a plasma kallikrein-binding protein can be developed include the human, mouse or rat plasma kallikrein amino acid sequence, one of these sequences, and 80%, 85%, 90%. , 95%, 96%, 97%, 98% or 99% identical, or fragments thereof, such as fragments of the sequences provided below.

The sequence of human plasma kallikrein used in the selection and subsequent screening of binding proteins is shown below (Accession No. NP_000883.2). The human plasma kallikrein (86 kDa) used was purified from human plasma by a commercial vendor and activated by Factor XIIa. Factor XIIa activates prekallikrein by cleaving the polypeptide sequence at a single site (between Arg371-Ile372, the cleavage site indicated by a "/" in the sequence below), resulting in an approximately 52 kDa heavy produces active plasma kallikrein consisting of two disulfide-linked polypeptides, a chain and a catalytic domain of approximately 34 kDa (Colman and Schmaier (1997) "Contact System: A Vascular Biology Modulator With Anticoagulant, Attributes" Blood, 90, 3819-3843).

The human, mouse and rat prekallikrein amino acid sequences and the mRNA sequences encoding them are set forth below. The sequence of prekallikrein is similar to that of plasma except that the active plasma kallikrein (pKal) has a single polypeptide chain cleaved at a single position (indicated by "/") to produce two chains. Identical to kallikrein. The sequence provided below is the complete sequence including the signal sequence. Upon secretion from the expressing cells, the signal sequence is expected to be removed.

Exemplary plasma kallikrein proteins from various species are NP_000883.2 (human pKal protein), NM_000892 (human pKal mRNA), NP_032481.1 (mouse pKal protein), NM_008455.2 (mouse pKal mRNA), NP_036857.2. (rat pKal protein) and NM_012725 (rat pKal mRNA) in the GeneBank.

(B) Kallikrein inhibitors Kunitz domain inhibitors. A number of useful inhibitors of either tissue and/or plasma kallikrein contain the Kunitz domain. Exemplary Kunitz domain inhibitors are described in US Patent Application Publication US20100183625, incorporated herein by reference.

As used herein, a "Kunitz domain" is a polypeptide domain having at least 51 amino acids and containing at least 2, preferably 3 disulfides. The domain folds so that cysteines 1 and 6, cysteines 2 and 4, and cysteines 3 and 5 form disulfide bonds (e.g., in the Kunitz domain with 58 amino acids, cysteines can be present at positions corresponding to amino acids 5, 14, 30, 38, 51 and 55, according to the BPTI homologous sequence numbering provided below, and the disulfides at positions 5 and 55, between cysteines at positions 14 and 38, and 30 and 51), or, if two disulfide bonds are present, form disulfide bonds between corresponding subsets of those cysteines. can be folded. The spacing between each cysteine is amino acids 7, 5, 4 at positions corresponding to positions 5 to 55, 14 to 38 and 30 to 51, according to the BPTI sequence numbering provided below. , 3, 2, 1 or 0. The BPTI sequence can be used as a reference to refer to specific positions in any generic Kunitz domain. A comparison of the Kunitz domain of interest with BPTI is performed by identifying the optimal alignment that maximizes the number of aligned cysteines.

The 3D structure (high resolution) of the Kunitz domain of BPTI is known. One of the X-ray structures has been deposited in the Brookhaven Protein Data Bank as "6PTI". The 3D structures of several BPTI homologues are known (Eigenbrot et al. (1990) Protein Engineering 3(7):591-598; Hynes et al. (1990) Biochemistry 29:10018-10022). At least 81 Kunitz domain sequences are known. The known human homologue is the three Kunitz domains of LACI, also known as tissue factor pathway inhibitor (TFPI) (Wun et al. (1988) J. Biol. Chem. 263(13):6001-6004; Girard (1989) Nature, 338:518-520; Novotny et al., (1989) J. Biol. (1988) J. Biol. Chem., 263(34):18104-18107), the Kunitz domain from collagen, the three Kunitz domains of TFPI-2 (Sprecher et al. (1994) PNAS USA, 91:3353-3357). ), Hepatocyte Growth Factor Activator Inhibitor Type I Kunitz Domain, Hepatocyte Growth Factor Activator Inhibitor Type II Kunitz Domain, Kunitz Domains described in US Patent Publication No. 2004-0152633. LACI is a human serum phosphoglycoprotein with a molecular weight of 39 kDa containing three Kunitz domains.

The Kunitz domains described above are called LACI-K1 (residues 50-107), LACI-K2 (residues 121-178), and LACI-K3 (residues 213-270). The cDNA sequence of LACI is reported by Wun et al. (J. Biol. Chem., 1988, 263(13):6001-6004). Girard et al. (Nature, 1989, 338:518-520) report mutation studies in which the P1 residue in each of the three Kunitz domains was altered. LACI-K1 inhibits Factor VIIa (F.VIIa) and LACI-K2 inhibits Factor Xa when it forms a complex with tissue factor.

Exemplary Kunitz domain-containing proteins include the following, with SWISS-PROT accession numbers in parentheses.
Ａ４＿ＨＵＭＡＮ（Ｐ０５０６７）、Ａ４＿ＭＡＣＦＡ（Ｐ５３６０１）、Ａ４＿ＭＡＣＭＵ（Ｐ２９２１６）、Ａ４＿ＭＯＵＳＥ（Ｐ１２０２３）、Ａ４＿ＲＡＴ（Ｐ０８５９２）、Ａ４＿ＳＡＩＳＣ（Ｑ９５２４１）、ＡＭＢＰ＿ＰＬＥＰＬ（Ｐ３６９９２）、ＡＰＰ２＿ＨＵＭＡＮ（Ｑ０６４８１）、ＡＰＰ２＿ＲＡＴ（Ｐ１５９４３）、ＡＸＰ１＿ＡＮＴＡＦ（Ｐ８１５４７）、 ＡＸＰ２＿ＡＮＴＡＦ（Ｐ８１５４８）、ＢＰＴ１＿ＢＯＶＩＮ（Ｐ００９７４）、ＢＰＴ２＿ＢＯＶＩＮ（Ｐ０４８１５）、ＣＡ１７＿ＨＵＭＡＮ（Ｑ０２３８８）、ＣＡ３６＿ＣＨＩＣＫ（Ｐ１５９８９）、ＣＡ３６＿ＨＵＭＡＮ（Ｐ１２１１１）、ＣＲＰＴ＿ＢＯＯＭＩ（Ｐ８１１６２）、ＥＬＡＣ＿ＭＡＣＥＵ（Ｏ６２８４５）、ＥＬＡＣ＿ＴＲＩＶＵ（Ｑ２９１４３）、ＥＰＰＩ＿ＨＵＭＡＮ（Ｏ９５９２５）、 ＥＰＰＩ＿ＭＯＵＳＥ（Ｑ９ＤＡ０１）、ＨＴＩＢ＿ＭＡＮＳＥ（Ｐ２６２２７）、ＩＢＰ＿ＣＡＲＣＲ（Ｐ００９９３）、ＩＢＰＣ＿ＢＯＶＩＮ（Ｐ００９７６）、ＩＢＰＩ＿ＴＡＣＴＲ（Ｐ１６０４４）、ＩＢＰＳ＿ＢＯＶＩＮ（Ｐ００９７５）、ＩＣＳ３＿ＢＯＭＭＯ（Ｐ０７４８１）、ＩＭＡＰ＿ＤＲＯＦＵ（Ｐ１１４２４）、ＩＰ５２＿ＡＮＥＳＵ（Ｐ１０２８０）、ＩＳＣ１＿ＢＯＭＭＯ（Ｐ１０８３１）、 ＩＳＣ２＿ＢＯＭＭＯ（Ｐ１０８３２）、ＩＳＨ１＿ＳＴＯＨＥ（Ｐ３１７１３）、ＩＳＨ２＿ＳＴＯＨＥ（Ｐ８１１２９）、ＩＳＩＫ＿ＨＥＬＰＯ（Ｐ００９９４）、ＩＳＰ２＿ＧＡＬＭＥ（Ｐ８１９０６）、ＩＶＢ１＿ＢＵＮＦＡ（Ｐ２５６６０）、ＩＶＢ１＿ＢＵＮＭＵ（Ｐ００９８７）、ＩＶＢ１＿ＶＩＰＡＡ（Ｐ００９９１）、ＩＶＢ２＿ＢＵＮＭＵ（Ｐ００９８９）、ＩＶＢ２＿ＤＡＢＲＵ（Ｐ００９９０）、 ＩＶＢ２＿ＨＥＭＨＡ（Ｐ００９８５）、ＩＶＢ２＿ＮＡＪＮＩ（Ｐ００９８６）、ＩＶＢ３＿ＶＩＰＡＡ（Ｐ００９９２）、ＩＶＢＢ＿ＤＥＮＰＯ（Ｐ００９８３）、ＩＶＢＣ＿ＮＡＪＮＡ（Ｐ１９８５９）、ＩＶＢＣ＿ＯＰＨＨＡ（Ｐ８２９６６）、ＩＶＢＥ＿ＤＥＮＰＯ（Ｐ００９８４）、ＩＶＢＩ＿ＤＥＮＡＮ（Ｐ００９８０）、ＩＶＢＩ＿ＤＥＮＰＯ（Ｐ００９７９）、ＩＶＢＫ＿ＤＥＮＡＮ（Ｐ００９８２）、 IVBK_DENPO (P00981), IVBT_ERIMA (P24541), IVBT_NAJNA (P20229), MCPI_MELCP （Ｐ８２９６８）、ＳＢＰＩ＿ＳＡＲＢＵ（Ｐ２６２２８）、ＳＰＴ３＿ＨＵＭＡＮ（Ｐ４９２２３）、ＴＫＤ１＿ＢＯＶＩＮ（Ｑ２８２０１）、ＴＫＤ１＿ＳＨＥＥＰ（Ｑ２９４２８）、ＴＸＣＡ＿ＤＥＮＡＮ（Ｐ８１６５８）、ＵＰＴＩ＿ＰＩＧ（Ｑ２９１００）、ＡＭＢＰ＿ＢＯＶＩＮ（Ｐ００９７８）、ＡＭＢＰ＿ＨＵＭＡＮ（Ｐ０２７６０）、ＡＭＢＰ＿ＭＥＲＵＮ（Ｑ６２５７７）、ＡＭＢＰ＿ＭＥＳＡＵ （Ｑ６０５５９）、ＡＭＢＰ＿ＭＯＵＳＥ（Ｑ０７４５６）、ＡＭＢＰ＿ＰＩＧ（Ｐ０４３６６）、ＡＭＢＰ＿ＲＡＴ（Ｑ６４２４０）、ＩＡＴＲ＿ＨＯＲＳＥ（Ｐ０４３６５）、ＩＡＴＲ＿ＳＨＥＥＰ（Ｐ１３３７１）、ＳＰＴ１＿ＨＵＭＡＮ（Ｏ４３２７８）、ＳＰＴ１＿ＭＯＵＳＥ（Ｑ９Ｒ０９７）、ＳＰＴ２＿ＨＵＭＡＮ（Ｏ４３２９１）、ＳＰＴ２＿ＭＯＵＳＥ（Ｑ９ＷＵ０３）、ＴＦＰ２＿ＨＵＭＡＮ (P48307), TFP2_MOUSE (O35536), TFPI_HUMAN (P10646), TFPI_MACMU (Q28864), TFPI_MOUSE (O54819), TFPI_RABIT (P19761), TFPI_RAT (Q02445), YN81_CAEEL (Q03610)

Various methods can be used to identify Kunitz domains from sequence databases. For example, the known amino acid sequence, consensus sequence or motif (e.g., ProSite motif) of a Kunitz domain can be compared against the GenBank sequence database (National Center for Biotechnology Information, National Institutes of Health, Bethesda, Md.) using, e.g., BLAST; You can search against the Pfam database of HMMs (Hidden Markov Models), eg, using default parameters for Pfam searches; against the SMART database; or against the ProDom database. For example, Pfam Release 9, Pfam Accession No. PF00014, provides a number of Kunitz domains and HMMs for Kunitz domain identification. A description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3):405-420, and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al. (1994) J. Am. Mol. Biol. 235:1501-1531; and Stultz et al. (1993) Protein Sci. 2:305-314. HMM's SMART database (Simple Modular Architecture Research Tool, EMBL, Heidelberg, Del.) is based on Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857 and Schultz et al. (2000) Nucl. Acids Res 28:231. The SMART database contains domains identified by profiling with hidden Markov models of the HMMer2 search program (R. Durbin et al. (1998) Biological sequence analysis: probabilistic models of proteins and nucleic acids. Cambridge University Press). Databases are also annotated and monitored. The ProDom protein domain database consists of an automated compilation of homologous domains (Corpet et al. (1999) Nucl. Acids Res. 27:263-267). The current version of ProDom supports recursive PSI-BLAST searches of the SWISS-PROT38 and TREMBL protein databases (Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402; Gouzy et al. (1999) Computers and Chemistry 23:333-340). is constructed using The database automatically generates consensus sequences for each domain. Prosite lists Kunitz domains as motifs and identifies proteins containing Kunitz domains. For example, Falquet et al. Nucleic Acids Res. 30:235-238 (2002).

Kunitz domains interact with target proteases primarily using amino acids in two loop regions (“binding loops”). The first loop region lies between approximately residues corresponding to amino acids 13-20 of BPTI. The second loop region is between approximately residues corresponding to amino acids 31-39 of BPTI. An exemplary library of Kunitz domains varies in one or more amino acid positions in the first and/or second loop regions. Particularly useful positions to change when screening for Kunitz domains that interact with kallikrein, or when selecting for affinity-improved variants are relative to the sequence of BPTI 13, 15, 16, 17, 18, Including the 19th, 31st, 32nd, 34th and 39th positions. At least some of these positions are predicted to be in close proximity to the target protease. It may be useful to vary other positions as well, such as positions adjacent to the above-mentioned positions in the three-dimensional structure.

The "framework region" of a Kunitz domain is part of the Kunitz domain, but residues in the first and second binding loop regions, ie, amino acids 13-20 of BPTI and amino acids 31-39 of BPTI. are defined as residues specifically excluding the approximate residues corresponding to . Conversely, residues that are not in the binding loop can tolerate a wide range of amino acid substitutions (eg, conservative and/or non-conservative substitutions).

In one embodiment, these Kunitz domains are loop-structure variants comprising Kunitz domain 1 of the human lipoprotein-associated inhibitor of coagulation (LACI) protein. LACI contains three internal distinct peptide loop structures, which are typical of Kunitz domains (Girard, T. et al., 1989. Nature, 338:518-520). The Kunitz domain 1 variants of LACI described herein have been screened, isolated, and bind kallikrein with improved affinity and specificity (e.g., U.S. Pat. No. 5,795). , 865 and US Pat. No. 6,057,287). These methods can also be applied to other Kunitz domain frameworks to obtain other Kunitz domains that interact with kallikrein, such as plasma kallikrein. Useful modulators of kallikrein function typically bind and/or inhibit kallikrein as determined using kallikrein binding and inhibition assays.

An exemplary Kunitz domain-containing polypeptide that inhibits plasma kallikrein has or comprises the amino acid sequence defined by amino acids 3-60 of SEQ ID NO:2. Another exemplary polypeptide comprising a Kunitz domain that inhibits plasma kallikrein has or comprises the amino acid sequence of SEQ ID NO:2.

An exemplary polypeptide has the amino acid sequence:
Ｘａａ１ Ｘａａ２ Ｘａａ３ Ｘａａ４ Ｃｙｓ Ｘａａ６ Ｘａａ７ Ｘａａ８ Ｘａａ９ Ｘａａ１０ Ｘａａ１１ Ｇｌｙ Ｘａａ１３ Ｃｙｓ Ｘａａ１５ Ｘａａ１６ Ｘａａ１７ Ｘａａ１８ Ｘａａ１９ Ｘａａ２０ Ｘａａ２１ Ｘａａ２２ Ｘａａ２３ Ｘａａ２４ Ｘａａ２５ Ｘａａ２６ Ｘａａ２７ Ｘａａ２８ Ｘａａ２９ Ｃｙｓ Ｘａａ３１ Ｘａａ３２ Ｐｈｅ Ｘａａ３４ Ｘａａ３５ Ｇｌｙ Ｇｌｙ Ｃｙｓ Ｘａａ３９ Ｘａａ４０ Ｘａａ４１ Ｘａａ４２ Ｘａａ４３ Ｘａａ４４ Ｘａａ４５ Ｘａａ４６ Ｘａａ４７ Ｘａａ４８ Ｘａａ４９ Ｘａａ５０ Cys Xaa52 Xaa53 Xaa54 Cys Xaa56 Xaa57 Xaa58 (SEQ ID NO: 1)
including.

"Xaa" refers to a position in a peptide chain that can be any of a number of different amino acids. In a first example, Xaa can be any amino acid except cysteine. In other examples, one or more of the following apply: Xaa10 can be Asp or Glu; Xaa11 can be Asp, Gly, Ser, VaI, Asn, Ile, Ala or Thr; Xaa13 can be Pro, Arg, His, Asn, Ser, Thr, Ala, Gly, Lys or Gln; Xaa15 can be Arg, Lys, Ala, Ser, Gly, Met, Asn or Gln; , Ala, Gly, Ser, Asp or Asn; Xaa17 can be Ala, Asn, Ser, Ile, Gly, Val, Gln, or Thr; Xaa18 can be His, Leu, Gln or Ala Xaa19 can be Pro, Gln, Leu, Asn or Ile; Xaa21 can be Trp, Phe, Tyr, His or He; Xaa31 can be Glu, Asp, Gln, Asn, Ser, Ala, Val, Xaa32 can be Glu, Gln, Asp, Asn, Pro, Thr, Leu, Ser, Ala, Gly or Val; Xaa34 can be Ile, Thr, Ser, Val, Ala, Xaa35 can be Tyr, Trp or Phe; Xaa39 can be Glu, Gly, Ala, Ser or Asp. Amino acids Xaa6, Xaa7, Xaa8, Xaa9, Xaa20, Xaa24, Xaa25, Xaa26, Xaa27, Xaa28, Xaa29, Xaa41, Xaa42, Xaa44, Xaa46, Xaa47, Xaa48, Xaa49, Xaa50, Xaa52, Xaa543 and any amino acid Xaa obtain.

Furthermore, each of the first four (Xaa1, Xaa2, Xaa3, Xaa4) and last three (Xaa56, Xaa57 or Xaa58) amino acids of SEQ ID NO: 1 may optionally be present or absent; If present, it can be any amino acid, including any non-cysteine amino acid.

In one embodiment, the polypeptide has a sequence with one or more of the following properties:
Xaa11 can be Asp, Gly, Ser or Val; Xaa13 can be Pro, Arg, His or Asn; Xaa15 can be Arg or Lys; Xaa16 can be Ala or Gly; , Ala, Asn, Ser or Ile; Xaa18 can be His, Leu or Gln; Xaa19 can be Pro, Gln or Leu; Xaa21 can be Trp or Phe; Xaa32 can be Glu or Gln; Xaa34 can be Ile, Thr or Ser; Xaa35 is Tyr; and Xaa39 can be Glu, Gly or Ala.

Exemplary polypeptides may contain the following amino acids:
Xaa10 is Asp; Xaa11 is Asp; Xaa13 can be Pro or Arg; Xaa15 is Arg; Xaa16 can be Ala or Gly; Xaa19 is Pro; Xaa21 is Trp; Xaa31 is Glu; Xaa32 is Glu; Xaa34 can be Ile or Ser; and Xaa39 is Gly.

It is also possible to use portions of the polypeptides described herein. For example, a polypeptide can include a binding domain for a specific kallikrein epitope. For example, the binding loops of Kunitz domains can be cyclized and used alone or can be grafted onto other domains, such as frameworks of other Kunitz domains. It is also possible to remove 1, 2, 3 or 4 amino acids from the N-terminus of the amino acid sequences described herein and/or the C of the amino acid sequences described herein. It is also possible to remove 1, 2, 3, 4 or 5 amino acids from the end.

Further examples of sequences are at least 1, but 7, 6, 5, 4, 3, for the amino acid sequences described herein, such as the amino acid sequences provided above. Includes sequences that differ by one or less than two amino acids. In one embodiment, 3, 2 or less than 1 differences are present in one of the binding loops. For example, the first binding loop can have no differences to the amino acid sequences described herein, such as the amino acid sequences provided above. In other embodiments, neither the first nor the second binding loop differ from the amino acid sequences described herein, such as the amino acid sequences provided above.

Still other polypeptides that inhibit plasma kallikrein include a PEP-1 polypeptide having a sequence of about 58 amino acids from amino acids 3-60 of SEQ ID NO:2 or a sequence of 60 amino acids of SEQ ID NO:2. . As used herein, both the terms “PEP-1” and “DX-88” refer to the 60 amino acid sequence of SEQ ID NO:2. In one embodiment, the polypeptide is other than aprotinin, eg, differs from aprotinin by at least 1, 2, 3, 5, 10 or 15 amino acids.

The polypeptides described herein can be made synthetically using any standard polypeptide synthesis protocol and equipment. For example, stepwise synthesis of a polypeptide involves removal of the amino (N)-terminal protecting group from the first (i.e., carboxy-terminal) amino acid and coupling to it the carboxyl-terminal of the next amino acid in the sequence of the polypeptide. can be implemented by Amino acids are also suitably protected. The carboxyl group of the resulting amino acid is activated to form a reactive group such as a carbodiimide, a symmetrical anhydride, or an "active ester" group such as a hydroxybenzotriazole or pentafluorophenyl ester to form a bound amino acid. It can be reacted with the N-terminus of the group. Preferred solid phase peptide synthesis methods are the BOC method using tert-butyloxycarbonyl as the I-amino protecting group and the FMOC method using the 9-fluorenylmethyloxycarbonyl group to protect the alpha-amino of the amino acid residue. including. Both methods are well known to those skilled in the art (Stewart, J. and Young, J., Solid-Phase Peptide Synthesis (WH Freeman Co., San Francisco 1989); Merrifield, J., 1963. Am. Chem. Soc., 85:2149-2154; Bodanszky, M. and Bodanszky, A., The Practice of Peptide Synthesis (Springer-Verlag, New York 1984)). If desired, additional amino- and/or carboxy-terminal amino acids can be designed to be included in the amino acid sequence and added during polypeptide synthesis.

Polypeptides can also be produced using recombinant techniques. Recombinant methods utilize any of a number of cell and corresponding expression vectors including, but not limited to, bacterial expression vectors, yeast expression vectors, baculovirus expression vectors, mammalian virus expression vectors, and the like. can. A polypeptide described herein can be produced by a transgenic animal, eg, in the mammary gland of the transgenic animal. In some cases, a sequence encoding a polypeptide that inhibits kallikrein (eg, a polypeptide comprising a Kunitz domain) is combined with other coding in an expression vector to form a fusion polypeptide that is readily expressed in a host cell. It may be necessary or beneficial to fuse the sequences. Part or all of the additional sequence can be removed, eg, by protease digestion.

An exemplary recombinant expression system for producing a polypeptide that inhibits kallikrein (e.g., a polypeptide comprising a Kunitz domain) is wherein the nucleic acid sequence encoding the amino acid sequence of the inhibitor polypeptide comprises the MATα prepro leader peptide sequence of Saccharomyces cerevisiae. Yeast expression vectors that are capable of being joined in the same reading frame as the encoding nucleotide sequence and are thereby under the control of a functional yeast promoter. The resulting recombinant yeast expression plasmid can be transformed into cells of a suitable compatible yeast host by standard methods, which are capable of expressing the recombinant protein from the recombinant yeast expression vector. Preferably, host yeast cells transformed with such recombinant expression vectors are also capable of processing the fusion protein to provide active inhibitor polypeptides. Another exemplary yeast host for producing recombinant polypeptides is Pichia pastoris.

As noted above, a polypeptide that inhibits kallikrein can include a Kunitz domain as described herein. Some polypeptides may include additional flanking sequences, preferably 1-6 amino acids in length, at the amino and/or carboxy termini, provided that such additional amino acids are described herein. does not significantly reduce kallikrein binding affinity or kallikrein inhibitory activity so as to render it unusable in the methods and compositions described in . Such additional amino acids may be added intentionally to allow expression of the polypeptide in a particular recombinant host cell, or to provide additional functions, such as to provide linkers with other molecules. can be added to provide affinity moieties for purification or to facilitate purification of the polypeptide. Preferably, the additional amino acids do not contain cysteines that can interfere with the disulfide bonds of the Kunitz domain.

An exemplary Kunitz domain polypeptide comprises the amino acid sequence of residues 3-60 of SEQ ID NO:2. When expressed and processed in a yeast fusion protein expression system (eg, based on the integrating expression plasmid pHIL-D2), such Kunitz domain polypeptides are expressed in S. cerevisiae. It retains an additional amino-terminal Glu-Ala2 peptide derived from a fusion with the S. cerevisiae MATα-prepro leader peptide sequence. When secreted from the yeast host cell, most of the leader peptide is processed from the fusion protein to yield a functional polypeptide having the amino acid sequence of SEQ ID NO:2 (referred to herein as "PEP-1"). .

Exemplary Kunitz domains, including for example SEQ ID NO: 1, correspond to a number of invariant positions, such as positions 5, 14, 30, 33, 38, 45, 51 and 55 according to the BPTI numbering convention. The position is cysteine. The spacing between these positions can vary within the allowable range within the Kunitz domain fold, such that, for example, three disulfide bonds are formed. 6, 7, 8, 9, 20, 24, 25, 26, 27, 28, 29, 41, 42, 44, 46, 47, 48, 49, 50, 52, 53 and 54, or these Other positions, such as positions corresponding to positions of can be any amino acid (including amino acids that are not genetically encoded). In particularly preferred embodiments, one or more amino acids correspond to those of the native sequence. In other embodiments, at least one variable position differs from that of the native sequence. In yet other preferred embodiments, each of the amino acids may be replaced individually or collectively with conservative or non-conservative amino acid substitutions.

Conservative amino acid substitutions replace an amino acid with another amino acid of similar chemical nature and may not affect protein function. Non-conservative amino acid substitutions replace an amino acid with another amino acid of dissimilar chemical nature. Examples of conservative amino acids include, eg, Asn→Gln, Arg→Lys and Ser→Thr. In preferred embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 of these amino acids , 15, 16, 17, 18, 19, 20, and/or 21, individually or collectively, in any combination, to correspond to the corresponding positions of SEQ ID NO:2. can be selected.

For example, positions 10, 11, 13, 15, 16, 17, 18, 19, 21, 22, 23, 31, 32, 34, 35, 39, 40, 43 and 45, or corresponding to those positions. Other positions, such as positions, can be any of a selected set of amino acids. For example, SEQ ID NO: 1 defines a set of possible sequences. Each member of this set includes, for example, cysteines at positions 5, 14, 30, 51 and 55 and , 32, 34, 35, 39, 40, 43 and 45 or any of the specified set of amino acids at positions corresponding to these positions. In preferred embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 of these amino acids , 15, 16, 17, 18 and/or 19 may be selected individually or collectively, in any combination, to correspond to the corresponding positions of SEQ ID NO:2. The polypeptide preferably has at least 80%, 85%, 90%, 95%, 97%, 98% or 99% identity to SEQ ID NO:2.

The comparison of sequences and determination of percent homology between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent homology of two amino acid sequences is determined using either the Blossom62 matrix or the PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6 or 4 and 1, 2, 3, 4 , an algorithm incorporated in the GAP program in the GCG software package, Needleman and Wunsch (1970), J. Am. Mol. Biol. 48:444-453. In yet another preferred embodiment, the percent homology between the two nucleic acid sequences is NWSgapdna. Determined using the GAP program in the GCG software package, using the CMP matrix and gap weights of 40, 50, 60, 70 or 80 and length weights of 1, 2, 3, 4, 5 or 6. A particularly preferred set of parameters (and which should be used when the practitioner does not know which parameters to apply to determine whether a molecule falls within the limits of homology) is a gap penalty of 12, a Blossum62 scoring matrix with a gap extension penalty and a frameshift gap penalty of 5.

Binding Protein Inhibitors In other embodiments, the inhibitor of kallikrein is a binding protein, such as an antibody. Exemplary binding proteins such as antibodies are described, for example, in PCT Publication WO2012/094587 and US Patent Application Publication US20100183625, both of which are incorporated herein by reference in their entirety.

In one aspect, the disclosure features a protein (eg, an isolated protein) that binds plasma kallikrein (eg, human plasma kallikrein) and comprises at least one immunoglobulin variable region. For example, the protein comprises heavy chain (HC) immunoglobulin variable domain sequences and/or light chain (LC) immunoglobulin variable domain sequences. The protein can bind to and inhibit plasma kallikrein, such as human plasma kallikrein.

The protein may comprise one or more of the following properties: (a) human CDRs or human framework regions, (b) CDRs of HC variable domains described herein and at least 85%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical CDRs with one or more (e.g., 1 (c) the CDRs of the LC variable domains described herein and at least 85%, 88%, 89%, 90%, 91%, LC comprising one or more (e.g., 1, 2 or 3) CDRs that are 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical immunoglobulin variable domain sequences, (d) LC variable domains (e.g., whole or in framework regions or in CDRs) described herein and at least 85%, 88%, 89%, 90%, 91%; LC immunoglobulin variable domain sequences that are 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical, (e) an HC variable domain described herein ( e.g., throughout or in framework regions or in CDRs) and at least 85%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , an HC immunoglobulin variable domain sequence that is 99% or 100% identical, (f) binds to an epitope bound by a protein described herein or competes for binding with a protein described herein , a protein, (g) a primate CDR or primate framework region, (h) differing from CDR1 of an HC variable domain described herein by at least one amino acid, but by more than two or three amino acids HC immunoglobulin variable domain sequences comprising CDR1 that do not differ, (i) differing by at least one amino acid from CDR2 of the HC variable domains described herein, but 2, 3, 4, 5 , an HC immunoglobulin variable domain sequence comprising a CDR2 that differs by no more than 6, 7 or 8 amino acids; (j) differs by at least 1 amino acid from the CDR3 of the HC variable domains described herein. more than 2, 3, 4, 5 or 6 (k) differing by at least one amino acid from the CDR1 of the LC variable domain described herein, but by 2, 3, 4; or an LC immunoglobulin variable domain sequence comprising a CDR1 that differs by no more than 5 amino acids, (l) differs by at least 1 amino acid from the CDR2 of the LC variable domains described herein, but by an LC immunoglobulin variable domain sequence comprising a CDR2 that differs by no more than one or four amino acids; an LC immunoglobulin variable domain sequence comprising a CDR3 that differs by no more than 3, 4 or 5 amino acids; LC immunoglobulin variables that differ by at least 1 amino acid from middle) but differ by no more than 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids the domain sequence, and (o) differing by at least one amino acid from the HC variable domains described herein (e.g., as a whole or in the framework regions or in the CDRs), but 2, 3, 4, 5 HC immunoglobulin variable domain sequences that differ by no more than 1, 6, 7, 8, 9 or 10 amino acids.

A plasma kallikrein-binding protein can be an isolated protein (eg, at least 70%, 80%, 90%, 95% or 99% free of other proteins). Plasma kallikrein-binding proteins can inhibit plasma kallikrein, such as human plasma kallikrein. In some embodiments, plasma kallikrein does not bind to prekallikrein (eg, human prekallikrein), but binds to the active form of plasma kallikrein (eg, human plasma kallikrein).

In certain embodiments, the protein binds at or near the active site of the catalytic domain of plasma kallikrein or a fragment thereof, or binds to an epitope overlapping the active site of plasma kallikrein. In some embodiments, the protein binds or competes for binding with the same epitope as a protein described herein.

In some embodiments, the protein is M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to herein as DX-2922), X81-B01, X67-D03, X67- G04, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X124-G01 (herein DX-2930), competes with or binds to the same epitope as X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04. See PCT Publication WO2010/094587 and US Patent Application Publication US20100183625.

In certain embodiments, the protein binds to one or more amino acids from the catalytic triad of plasma kallikrein: His434, Asp483 and/or Ser578 (numbering is based on the human sequence).

In some embodiments, the protein binds to one or more of Ser479, Tyr563 and/or Asp585 (numbering is based on the human sequence).

The protein is plasma kallikrein, eg human plasma kallikrein, and 10 ⁵ M ⁻¹ , 10 ⁶ M ⁻¹ , 10 ⁷ M ⁻¹ , 10 ⁸ M ⁻¹ , 10 ⁹ M ⁻¹ , 10 ¹⁰ M ⁻¹ and 10 It can bind with a binding affinity of ¹¹ M ⁻¹ . In certain embodiments, the protein binds human plasma kallikrein with a K _off of 1×10 ⁻³ s ⁻¹ , 5×10 ⁻⁴ s ⁻¹ or less than 1×10 ⁻⁴ s ⁻¹ . In certain embodiments, the protein binds plasma kallikrein but does not bind tissue kallikrein and/or plasma prekallikrein (e.g., the protein binds tissue kallikrein and/or plasma kallikrein less efficiently than it binds plasma kallikrein). Binds prekallikrein (eg, binds 5-fold, 10-fold, 50-fold, 100-fold, or 1000-fold less efficiently than a negative control, or does not bind at all).

In certain embodiments, the protein inhibits human plasma kallikrein activity with a Ki of less than 10 ⁻⁵ M, 10 ⁻⁶ M, 10 ⁻⁷ M, 10 ⁻⁸ M, 10 ⁻⁹ M, and 10 ⁻¹⁰ M . The protein may, for example, have an IC50 of less than 100 nM, 10 nM or 1 nM. For example, the protein can modulate plasma kallikrein activity and production of activated factor XII (eg, from factor XII) and/or production of bradykinin (eg, from high molecular weight kininogen (HMWK)). The protein can inhibit plasma kallikrein activity and/or the production of activated factor XII (eg from factor XII) and/or the production of bradykinin (eg from high molecular weight kininogen (HMWK)). The affinity of the protein for human plasma kallikrein can be characterized by a _Kd of less than 100 nM, less than 10 nM, or less than 1 nM. In certain embodiments, the protein inhibits plasma kallikrein but not tissue kallikrein (e.g., the protein inhibits tissue kallikrein with a lower efficiency than it inhibits plasma kallikrein (e.g., compared to a negative control). 5-fold, 10-fold, 50-fold, 100-fold or 1000-fold less efficient or no inhibition at all)).

In some embodiments, the protein has an apparent inhibition constant (K _i,app ) of less than 1000 nM, 500 nM, 100 nM or 10 nM.

A plasma kallikrein binding protein may be an antibody. Plasma kallikrein-binding antibodies can include the HC and LC variable domain sequences in a single polypeptide (eg scFv) or on different polypeptides (eg IgG or Fab).

In a preferred embodiment, the protein is M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to herein as DX-2922), X81-B01, X67-D03, X67- G04, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X124-G01 (herein DX-2930), X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04 (e.g., a human antibody) having light and/or heavy chains of an antibody selected from the group consisting of is.

In a preferred embodiment, the protein is M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to herein as DX-2922), X81-B01, X67-D03, X67- G04, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X124-G01 (herein DX-2930), one or more heavy chain CDRs selected from the corresponding CDRs of the heavy chain group consisting of X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04 ( eg, 1, 2 or 3) antibodies (eg, human antibodies).

In a preferred embodiment, the protein is M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to herein as DX-2922), X81-B01, X67-D03, X67- G04, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X124-G01 (herein DX-2930), one or more light chain CDRs selected from the corresponding CDRs of the group of light chains consisting of X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04 ( eg, 1, 2 or 3) antibodies (eg, human antibodies).

In a preferred embodiment, the protein is M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to herein as DX-2922), X81-B01, X67-D03, X67- G04, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X124-G01 (herein DX-2930), one or more heavy chain CDRs selected from the corresponding CDRs of the light chain group consisting of X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04 ( 1, 2 or 3) as well as antibodies (eg human antibodies) with one or more (eg 1, 2 or 3) light chain CDRs.

In some embodiments, the HC and LC variable domain sequences are components of the same polypeptide chain. In other embodiments, the HC and LC variable domain sequences are components of different polypeptide chains. For example, the protein is IgG, such as IgG1, IgG2, IgG3 or IgG4. The protein can be a soluble Fab. In other implementations, the protein is Fab2', scFv, minibody, scFv::Fc fusion, Fab::HSA fusion, HSA::Fab fusion, Fab::HSA::Fab fusion, or It includes other molecules that contain the antigen binding site of one of the binding proteins described herein. The VH and VL regions of these Fabs are IgG, Fab, Fab2, Fab2′, scFv, PEGylated Fab, PEGylated scFv, PEGylated Fab2, VH::CH1::HSA+LC, HSA::VH::CH1+LC, LC ::HSA+VH::CH1, HSA::LC+VH::CH1 or other suitable structures.

In one embodiment, the protein is a human or humanized antibody or non-immunogenic in humans. For example, the protein has one or more human antibody framework regions, such as all human framework regions, or at least 85%, 88%, 89%, 90%, 91%, 92% human framework regions. , 93%, 94%, 95%, 96%, 97%, 98%, 99% identical framework regions. In one embodiment, the protein comprises a human Fc domain or an Fc domain that is at least 95%, 96%, 97%, 98% or 99% identical to a human Fc domain.

In one embodiment, the protein is a primate or primatizing antibody, or is non-immunogenic in humans. For example, the protein may be at least 85%, 88%, 89%, 90% combined with one or more primate antibody framework regions, such as all primate framework regions, or primate framework regions. , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical framework regions. In one embodiment, the protein comprises a primate Fc domain or an Fc domain that is at least 95%, 96%, 97%, 98% or 99% identical to a primate Fc domain. "Primates" include humans (Homo sapiens), chimpanzees (Pan troglodytes and Pan paniscus (bonobos)), gorillas (Gorilla gorilla), gibbons, monkeys, lemurs, aye-ayes (Daubentonia madagascariensis) and tarsiers.

In certain embodiments, the affinity of the primate antibody for human plasma kallikrein is characterized by a K _D of less than 1000 nM, 500 nM, 100 nM or 10 nM, such as less than 10 nM or less than 1 nM.

In one embodiment, the protein comprises a human framework region or a framework region that is at least 95%, 96%, 97%, 98% or 99% identical to a human framework region. In some embodiments, the protein does not contain any mouse or rabbit derived sequences (eg, it is not a mouse or rabbit antibody).

In certain embodiments, the present disclosure provides proteins (e.g., binding proteins such as antibodies) that bind plasma kallikrein (e.g., human plasma kallikrein) and comprise at least one immunoglobulin variable region (e.g., binding proteins such as antibodies described herein). proteins) in methods of treating the diseases or disorders described herein. For example, a plasma kallikrein-binding protein comprises a heavy chain (HC) immunoglobulin variable domain sequence and a light chain (LC) immunoglobulin variable domain sequence. A number of exemplary plasma kallikrein-binding proteins are described herein.

Antibodies can be discovered by screening libraries with kallikrein targets, as well as by other methods. For example, kallikrein proteins or regions thereof can be used as antigens in non-human animals, such as rodents. Humanized antibodies can be produced by replacing sequences of the Fv variable region not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for producing humanized antibodies are described by Morrison, S.; L. , 1985, Science 229:1202-1207; Oi et al., 1986, BioTechniques 4:214; and Queen et al., US Pat. , 693,762. Those methods involve isolating, manipulating, and expressing nucleic acid sequences encoding all or part of an immunoglobulin Fv variable region from at least one of a heavy or light chain. Numerous sources of such nucleic acids are available. For example, nucleic acids can be obtained from hybridomas producing antibodies against a given target as described above. Recombinant DNA encoding the humanized antibody, or fragment thereof, can then be cloned into an appropriate expression vector.

Immunoglobulin kallikrein binding proteins (eg, IgG or Fab kallikrein binding proteins) can be modified to reduce immunogenicity. Reduced immunogenicity is desirable for kallikrein-binding proteins intended for use as therapeutics to reduce the likelihood that a subject will mount an immune response to the therapeutic molecule. Techniques useful for reducing the immunogenicity of kallikrein-binding proteins include removal/modification of potential human T-cell epitopes and "germlining" of sequences outside the CDRs (e.g., framework and Fc). include.

Kallikrein-binding antibodies can be modified by specific removal or "deimmunization" of human T-cell epitopes by the methods disclosed in WO98/52976 and WO00/34317. Briefly, antibody heavy and light chain variable regions are analyzed for peptides that bind MHC class II (as defined in WO98/52976 and WO00/34317) that represent potential T cell epitopes. A computer modeling approach termed "peptide threading" can be applied for the detection of potential T cell epitopes, as described in WO98/52976 and WO00/34317, and a database of human MHC class II binding peptides can be applied. can be searched for motifs present in VH and VL sequences. These motifs bind any of the 18 major MHC class IIDR allotypes and thus constitute potential T-cell epitopes. Potential T-cell epitopes detected can be eliminated by substituting small numbers of amino acid residues, preferably single amino acid substitutions, in the variable region. Often, but not limited to, amino acids common to that position in human germline antibody sequences may be used as long as possible conservative substitutions are made. Human germline sequences can be found in Tomlinson, I.; A et al., 1992, J. Am. Mol. Biol. 227:776-798; Cook, G.; P. et al., 1995, Immunol. Today Vol. 16(5) 237-242; Chothia, D.; et al., 1992, J. et al. Mol. Bio. 227:799-817. The V BASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (compiled by Tomlinson, IA et al., MRC Center for Protein Engineering, Cambridge, UK). After identification of deimmunizing alterations, nucleic acids encoding _VH and _VL can be constructed by mutagenesis or other synthetic methods (eg, de novo synthesis, cassette exchange, etc.). The mutagenized variable sequences can optionally be fused to human constant regions, such as IgG1 or kappa constant regions.

In some cases, potential T-cell epitopes will include residues known or predicted to be important for antibody function. For example, potential T cell epitopes usually have a bias towards CDRs. In addition, potential T-cell epitopes can occur in framework residues important for antibody structure and binding. Modifications to eliminate these potential pitopes sometimes require more scrutiny, eg, making and testing strands with and without modifications. Where possible, potential T-cell epitopes overlapping the CDRs are eliminated by substitutions outside the CDRs. In some cases, an alteration within a CDR is the only option, so variants with and without this substitution should be tested. In other cases, substitutions required to remove potential T-cell epitopes occur at residue positions within the framework that may be important for antibody binding. In these cases, variants with and without this substitution should be tested. Therefore, in some cases, several variant deimmunized heavy and light chain variable regions were designed and various heavy/light chain combinations were tested to identify the optimal deimmunized antibody. The final deimmunized antibody selection is made by considering the binding affinities of the different variants together with the degree of deimmunization, i.e. the number of potential T-cell epitopes left in the variable region. . Deimmunization can be used to modify any antibody, including antibodies that contain non-human sequences, such as synthetic antibodies, murine antibodies, other non-human monoclonal antibodies, or antibodies isolated from display libraries. .

Kallikrein-binding antibodies are "germinated" by converting one or more non-germline amino acids in the framework regions to the corresponding germline amino acids of the antibody, so long as binding capacity remains substantially intact. cell lineage”. Similar methods are also available for constant regions, such as immunoglobulin constant domains.

Antibodies that bind kallikrein, such as the antibodies described herein, can be modified to make the variable regions of the antibody more similar to one or more of the germline sequences. For example, an antibody may contain one, two, three or more amino acid substitutions, eg, in the framework, CDRs or constant regions, to make it more similar to the reference germline sequence. One exemplary germlining method involves identifying one or more germline sequences that are similar (e.g., most similar in a particular database) to the isolated antibody sequence. can contain. Mutations (at the amino acid level) are then made in the isolated antibody either incrementally or in combination with other mutations. For example, a nucleic acid library containing sequences encoding some or all possible germline mutations is generated. A mutated antibody is then prepared, e.g., an antibody that has one or more additional germline residues compared to the isolated antibody, but is still useful (e.g., has functional activity). Evaluate for identification. In one embodiment, as many germline residues as possible are introduced into the isolated antibody.

In one embodiment, mutagenesis is used to substitute or insert one or more germline residues in the framework and/or constant regions. For example, the germline framework and/or constant region residues can be derived from germline sequences that are similar (eg, most similar) to the non-variable region to be modified. After mutagenesis, activity (eg, binding or other functional activity) of the antibody can be assessed to determine whether the germline residue or residues are tolerated. Similar mutations can be made in framework regions.

Selection of germline sequences can be performed by different methods. For example, germline sequences may have a predetermined criterion for selectivity or similarity, e.g., at least a specified percent identity, e.g. %, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identity. Selection can be performed using at least 2, 3, 5 or 10 germline sequences. In the case of CDR1 and CDR2, identification of similar germline sequences may involve selection of one such sequence. In the case of CDR3, identification of similar germline sequences may involve selection of one such sequence, but two germline sequences contributing to the amino-terminal and carboxy-terminal portions separately. can include using In other implementations, more than one or two germline sequences are used, eg, to form a consensus sequence.

In one embodiment, for a particular reference variable domain sequence, such as the sequences described herein, the related variable domain sequence has at least 30% of CDR amino acid positions that are not identical to residues in the reference CDR sequence. , 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% and corresponding in the human germline sequence (i.e., the amino acid sequence encoded in the human germline nucleic acid) It has residues that are identical to the residues in the position.

In one embodiment, for a particular reference variable domain sequence, eg, a sequence described herein, the related variable domain sequence is a human germline sequence, eg, a germline sequence related to the reference variable domain sequence. Having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% FR regions identical to the FR sequences derived from the cell lineage sequence.

Thus, antibodies are isolated that have similar activity to a given antibody of interest, but are more similar to one or more germline sequences, particularly one or more human germline sequences. It is possible to For example, the antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% germline sequence outside of the CDRs (e.g., framework regions). , 99% or 99.5% identical. Furthermore, the antibody has at least 1, 2, 3, 4 or 5 germline sequences in the CDR regions that are derived from similar (e.g., most similar) germline sequences to the variable regions to be modified. may contain residues of The germline sequences of most interest are human germline sequences. Antibody activity (e.g. avidity as measured by KA) is 1 _- fold or 100-fold, 10-fold, 5-fold, 2-fold, 0.5-fold, 0.1-fold and 0.001-fold that of the original antibody can be in the range of

The germline sequences of the human immunoglobulin genes have been determined and are available through the world wide web at imgt. cines. International ImMunoGeneTics information system (IMGT) available at fr, and the V BASE Directory (MRC Center for Protein Engineering, Cambridge, UK, edited by Tomlinson, IA et al., vbase.mrc through the World Wide Web). - available from a number of sources, including available at cpe.cam.ac.uk).

Exemplary germline reference sequences for _Vkappa are: O12/O2, O18/O8, A20, A30, L14, L1, L15, L4/18a, L5/L19, L8, L23, L9, L24, L11 , L12, O11/O1, A17, A1, A18, A2, A19/A3, A23, A27, A11, L2/L16, L6, L20, L25, B3, B2, A26/A10 and A14. See, eg, Tomlinson et al., 1995, EMBO J.; 14(18):4628-3.

Germline reference sequences for HC variable domains can be based on sequences with specific canonical structures, eg, 1-3 structures in the hypervariable loops of H1 and H2. Canonical structures of hypervariable loops of immunoglobulin variable domains are described in Chothia et al., 1992, J. Am. Mol. Biol. 227:799-817; Tomlinson et al., 1992, J. Am. Mol. Biol. 227:776-798 and Tomlinson et al., 1995, EMBO J. et al. 14(18):4628-38, it can be deduced from its sequence. Exemplary sequences having 1-3 structures are: DP-1, DP-8, DP-12, DP-2, DP-25, DP-15, DP-7, DP-4, DP-31, DP-32, DP-33, DP-35, DP-40, 7-2, hv3005, hv3005f3, DP-46, DP-47, DP-58, DP-49, DP-50, DP-51, DP- 53 and DP-54.

Useful polypeptides can also be encoded by nucleic acids that hybridize to nucleic acids encoding the polypeptides described herein. The nucleic acids can hybridize under moderately stringent, highly stringent, or very highly stringent conditions. As used herein, the term "hybridize under low, moderate, high or very high stringency conditions" describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.W. Y. (1989), 6.3.1-6.3.6. can be found in Aqueous and non-aqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are as follows: (1) in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by at least 50° C. (under low stringency conditions); (2) low stringency hybridization conditions with two washes in 0.2 x SSC, 0.1% SDS at about 45°C in 6 x SSC; (3) stringent hybridization conditions in 0.2×SSC at 60° C., followed by one or more washes in 0.1% SDS; (3) in 6×SSC at about 45° C.; , high stringency hybridization conditions with one or more washes in 0.2×SSC, 0.1% SDS at 65° C., and (4) very high stringency hybridization conditions, 0.5 M sodium phosphate, 7% SDS at 65°C, followed by one or more washes in 0.2 x SSC, 1% SDS at 65°C.

protein production. Proteins that bind plasma kallikrein can be expressed using standard recombinant nucleic acid methods. Generally, a nucleic acid sequence encoding the protein is cloned into a nucleic acid expression vector. Of course, if the protein comprises multiple polypeptide chains, each chain can be cloned in expression vectors to be expressed in the same or different cells, eg the same or different vectors.

Antibody production. Some antibodies, such as Fab, have been produced by, for example, E. It can be produced in bacterial cells such as cells of E. coli. For example, if the Fab is encoded by a sequence in a phage display vector that includes a suppressible stop codon between the display moiety and the bacteriophage protein (or fragment thereof), the vector's nucleic acid is a bacterium that cannot suppress the stop codon. It can be transfected into cells. In this case the Fab is not fused to the geneIII protein and is secreted into the periplasm and/or medium.

Antibodies can also be produced in eukaryotic cells. In one embodiment, antibodies (eg, scFv) are expressed in yeast cells such as Pichia (see, eg, Powers et al., 2001, J. Immunol. Methods. 251:123-35), Hanseula, or Saccharomyce.

In one preferred embodiment, antibodies are produced in mammalian cells. A preferred mammalian host cell for expressing a cloned antibody or antigen-binding fragment thereof is the Chinese Hamster Ovary (CHO cell) (eg, DHFR as described in Kaufman and Sharp, 1982, Mol. Biol. 159:601-621). Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. Included are spherical cell lines, COS cells, HEK293T cells (Immunol. Methods (2004) 289(1-2):65-80), and cells derived from transgenic animals such as transgenic mammals. For example, the cells are mammary epithelial cells.

In addition to the nucleic acid sequence encoding the diversified immunoglobulin domain, the recombinant expression vector contains additional sequences such as sequences that regulate replication of the vector in a host cell (eg, origins of replication) and sequences for selectable marker genes. can have The selectable marker gene facilitates selection of host cells into which the vector has been introduced (e.g., US Pat. Nos. 4,399,216, 4,634,665 and 5,179,017). ). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr ^- host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).

In an exemplary system for recombinant expression of antibodies or antigen-binding portions thereof, recombinant expression vectors encoding both antibody heavy and light chains are introduced into dhfr ^- CHO cells by calcium phosphate-mediated transfection. . Within the recombinant expression vector, the antibody heavy and light chain genes are combined with enhancer/promoter regulatory elements (such as the CMV enhancer/AdMLP promoter regulatory element or the SV40 enhancer/AdMLP promoter) to drive high levels of transcription of the genes. (derived from SV40, CMV, adenovirus, etc.), such as regulatory elements, respectively). The recombinant expression vector also has a DHFR gene to allow selection of CHO cells transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are cultured to permit expression of antibody heavy and light chains, and intact antibody is recovered from the medium. Standard molecular biology techniques are used for preparing recombinant expression vectors, transfecting host cells, selecting transformants, culturing host cells and recovering antibody from the culture medium. For example, some antibodies are isolated by affinity chromatography with protein A or protein G binding matrices.

For antibodies that include an Fc domain, antibody production systems can produce antibodies that are glycosylated in the Fc region. For example, the Fc domain of IgG molecules is glycosylated at asparagine 297 of the CH2 domain. This asparagine is the site of modification with biantennary-type oligosaccharides. It has been demonstrated that this glycosylation is required for effector functions mediated by Fcg receptors and complement C1q (Burton and Woof, 1992, Adv. Immunol. 51:1-84; Jefferis et al., 1998). , Immunol. Rev. 163:59-76). In one embodiment, the Fc domain is produced in a mammalian expression system that appropriately glycosylate the residue corresponding to asparagine 297. The Fc domain may also contain other eukaryotic post-translational modifications.

Antibodies can also be produced in transgenic animals. For example, US Pat. No. 5,849,992 describes methods of expressing antibodies in the mammary gland of transgenic mammals. A transgene is constructed containing a milk-specific promoter and nucleic acid encoding the antibody of interest, as well as a signal sequence for secretion. The milk produced by females of such transgenic mammals contains the antibody of interest secreted therein. The antibody can be purified from the milk or, in some applications, used directly.

(C) Modifications Polypeptides that inhibit kallikrein can be modified in a variety of ways. For example, to stabilize the compound or to prolong the retention time, for example, by at least 2-fold, 4-fold, 5-fold, 8-fold, 10-fold, 15-fold, 20-fold, 50-fold, 100-fold, 500-fold or 1000-fold For this purpose, the polypeptide can be conjugated to one or more polyethylene glycol moieties.

In one embodiment, the kallikrein-binding protein is stabilized and/or retained in circulation, e.g., in blood, serum, lymph, or in other tissues, e.g., at least 1.5-fold, 2-fold, 5-fold , with moieties that enhance 10-fold or 50-fold. For example, a kallikrein-binding protein can be conjugated to a polymer, such as a substantially non-antigenic polymer such as a polyalkylene oxide or polyethylene oxide. Suitable polymers will vary substantially by weight. Polymers with number average molecular weights in the range of about 200 to about 35,000 (or about 1,000 to about 15,000 and 2,000 to about 12,500) can be used. For example, kallikrein-binding proteins can be conjugated to water-soluble polymers, such as hydrophilic polyvinyl polymers such as polyvinyl alcohol and polyvinylpyrrolidone. Multiple polymer moieties, eg, at least 2, 3 or 4 such moieties having an average molecular weight of 2,000 to 7,000 daltons, can be attached to a single polypeptide. A non-limiting list of such polymers is polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycol, polyoxyethylenated polyols, copolymers thereof, and block copolymers, as long as the water solubility is maintained. Including block copolymers thereof.

For example, polypeptides can be conjugated to water-soluble polymers such as hydrophilic polyvinyl polymers such as polyvinyl alcohol and polyvinylpyrrolidone. A non-limiting list of such polymers is polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycol, polyoxyethylenated polyols, copolymers thereof, and block copolymers, as long as the water solubility is maintained. Including block copolymers thereof. Further useful polymers are polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene (Pluronics); polymethacrylates; carbomers; lactose, amylopectin, starch, hydroxyethyl starch. , amylase, dextran sulfate, dextran, dextrin, glycogen, or homopolysaccharides and heteropolysaccharides such as polysaccharide subunits of acid mucopolysaccharides such as hyaluronic acid. - and L-galactose, fucose, fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannuronic acid (for example polymannuronic acid or alginic acid), D-glucosamine, D- Branched and unbranched polysaccharides including galactosamine, D-glucose and neuraminic acid; polymers of sugar alcohols such as polysorbitol and polymannitol; heparin or heparan.

One or more framework and/or CDR amino acid residues of the binding protein have one or more mutations (e.g., substitutions (e.g., conservative substitutions or non essential amino acid substitutions), insertions or deletions). The plasma kallikrein binding protein has a mutation (e.g., substitution (e.g., conservative or non-essential amino acid substitution), insertion or deletion) (e.g., at least 1, 2) compared to the binding protein described herein. , 3, or 5 and/or less than 15, 12, 10, 9, 8, 7, 6, 5, 4, 3 or 2 mutations), For example, it may have mutations that do not substantially affect protein function. Mutations can be in framework regions, CDRs and/or constant regions. In some embodiments, the mutation is in a framework region. In some embodiments the mutation is in a CDR. In some embodiments the mutation is in the constant region. Whether a particular substitution is tolerated, i.e., does not negatively affect biological properties such as binding activity, can be determined, for example, by assessing whether the mutation is conservative or by Bowie et al. (1990). Science 247:1306-1310.

A kallikrein binding protein can also be conjugated to a carrier protein such as serum albumin, eg human serum albumin. For example, protein fusions can be used to join carrier proteins and kallikrein-binding proteins.

(D) treatment of autoimmune diseases associated with the kallikrein system one or more of the pKal inhibitors described herein diabetic macular edema, retinal hyperplasia, brain trauma, acute spinal cord injury, focal amyloidosis , psoriasis, multiple sclerosis, inflammatory colitis, rheumatoid arthritis, vasculitis, autoimmune diseases such as systemic lupus erythematosus nephritis, systemic mastocytosis, severe burns, and neuropathic pain (diabetes and postherpetic neuralgia) ) for the treatment of diseases associated with the pKal system, including but not limited to.

A subject (e.g., a human patient) at risk of developing an autoimmune disease or other pKal-associated disease described herein includes, for example, a subject having a disease associated with the development of the disease, a subject Subjects may be exposed to relevant environmental factors, have a familial history of the disease, or have genes associated with the development of the disease.

The subject may be a human (e.g., a human having a disease or at risk of developing a disease) or a non-human subject (e.g., an autoimmune disease or other pKal-related disease) in need of treatment for one of the autoimmune diseases or other pKal-related diseases. (an animal model of pKal-related disease in ).

In certain embodiments, the subject is a subject (eg, a human patient) at risk of developing an autoimmune disease such as rheumatoid arthritis (RA), Crohn's disease (CD) or ulcerative colitis (UC). Autoimmune diseases are diseases caused by an abnormal immune response against the subject's own body. Aberrant immune responses may be directed against specific organs or tissues, depending on the type of autoimmune disease.

RA is a chronic inflammatory disease that generally affects joints such as the synovial joints of the hands and/or feet. The inflammatory response in RA often causes cartilage destruction and joint fusion, resulting in loss of function and mobility. Symptoms of RA include swollen and/or hot joints, stiffness, rheumatoid nodules, fatigue, fever, and weight loss. Exemplary treatments for RA include physical therapy, orthopedics, analgesics, anti-inflammatory drugs, steroids, and disease-modifying anti-rheumatic drugs (DMARDs).

CD is an inflammatory bowel disease that can affect any part of the gastrointestinal tract. Symptoms include abdominal pain, diarrhea, fever, malaise and weight loss. Exemplary treatments for CD include corticosteroids, 5-aminosalicylate drugs, azathioprine, methotrexate, infliximab, adalimumab, certolizumab, natalizumab, dietary modifications, and surgery.

UC is an inflammatory bowel disease that generally affects the large intestine. Symptoms include bloody and/or mucus-containing diarrhea, weight loss, anemia, abdominal pain, and rectal bleeding. Exemplary treatments for UC include 5-aminosalicylate drugs, corticosteroids, azathioprine, budesonide, infliximab, adalimumab, and surgery.

(E) Combination Therapy A plasma kallikrein inhibitor can be administered with other therapeutic agents as part of a combination therapy for the diseases or disorders described herein.

Combination therapy of a kallikrein inhibitor and other therapeutic agents can be provided in a number of different forms. In situations where the kallikrein inhibitor is administered as an intra-articular injection, the kallikrein inhibitor and therapeutic agent can be co-administered as a single composition or they can be administered by separate injections. In some situations, the kallikrein inhibitor and therapeutic agent are administered in close temporal proximity (e.g., within a short time interval, such as during the same treatment session), or the two components of a combination therapy are administered together. More widely spaced doses are given depending on the desired dosing schedule. When the kallikrein inhibitor is administered systemically (orally), the kallikrein inhibitor and the therapeutic agent are in close temporal proximity or closer together, depending on the intended dosing schedule for the two components of the combination therapy. It can be administered at wide intervals.

In other embodiments, kallikrein inhibitors may be administered in combination with other compounds useful for treating or preventing inflammation involved in autoimmune diseases. Exemplary anti-inflammatory agents include, for example, steroids (eg cortisol, cortisone, fludrocortisone, prednisone, 6[α]-methylprednisone, triamcinolone, betamethasone or dexamethasone), non-steroidal anti-inflammatory drugs (NSAIDs such as aspirin, acetaminophen, tolmetine, ibuprofen, mefenamic acid, piroxicam, nabumetone, rofecoxib, celecoxib, etodolac or nimesulide)). In other embodiments, the other therapeutic agent is an antibiotic (eg, vancomycin, penicillin, amoxicillin, ampicillin, cefotaxime, ceftriaxone, cefixime, rifampinmetronidazole, doxycycline, or streptomycin). In other embodiments, the other therapeutic agent is a PDE4 inhibitor (eg, roflumilast or rolipram). In other embodiments, the other therapeutic agent is an antihistamine (eg, cyclizine, hydroxyzine, promethazine, or diphenhydramine).

Further examples of anti-inflammatory agents are e.g. aceclofenac, acemethacin, e-acetamidocaproic acid, acetaminophen, acetaminosalol, acetanilide, acetylsalicylic acid, S-adenosylmethionine, alclofenac, alclomethasone, alfentanil, algestone, allylprozin. , aluminoprofen, alloxypyrine, alphaprozin, aluminum bis(acetylsalicylic acid), amcinonide, amphenac, aminochlortenoxazine, 3-amino-4-hydroxybutyric acid, 2-amino-4-picoline, aminopropyrone, aminopyrine, amixetrin, ammonium salicylate, ampiroxicam, amtolmethingacil, anileridine, antipyrine, anthraphenine, apazone, beclomethasone, bendazac, benolilate, benoxaprofen, benzpiperone, benzydamine, benzylmorphine, vermoprofen, betamethasone, betamethasone- 17-valeric acid, vezitramide, [α]-bisabolol, bromfenac, p-bromoacetanilide, 5-bromosalicylic acid acetate, bromosaligenin, busetin, bucloxic acid, bucolome, budesonide, bufexamac, bumazone, buprenorphine, butacetin, butibfen, butorphanol, carbamazepine , carbifene, carprofen, calsalam, chlorobutanol, chloroprednisone, chlortenoxazine, choline salicylate, cinchophene, simmethacin, cilamadol, clidanac, clobetasol, clocortolone, clomethacin, clonitazen, clonyxin, clopyrac, cloprednol, clove, codeine, codeine Including methyl bromide, codeine phosphate, codeine sulfate, cortisone, cortibazole, clopropamide, crotetamide and cyclazocine.

Further examples of anti-inflammatory agents are deflazacort, dehydrotestosterone, desomorphine, desonide, desoxymethasone, dexamethasone, dexamethasone-21-isonicotinic acid, dexoxadrol, dextromoramide, dextropropoxyphene, deoxycorticosterone. , Dezocine, Diampromide, Diamorphone, Diclofenac, Difenamisole, Difenpyramide, Diflorazone, Diflucortolone, Diflunisal, Difluprednate, Dihydrocodeine, Dihydrocodeine Nonenol Acetate, Dihydromorphine, Dihydroxyaluminum Acetylsalicylate, Dimenoxadol, Dimefe Butanol, dimethylthiambutene, dioxafetyl butyrate, dipipanone, diprocetyl, dipyrone, ditazol, droxicam, emmorphazone, enfenamic acid, enoxolone, epirizole, eptazocin, ethersalate, ethenzamide, ethoheptadine, ethoxazene, ethylmethylthiambutene, ethyl Morphine, Etodolac, Etofenamate, Etonitazene, Eugenol, Felbinac, Fenbufen, Fenclodic Acid, Fendozal, Fenoprofen, Fentanyl, Fentiazac, Feprazinol, Feplazone, Floctafenin, Fluazacort, Fluchloronide, Flufenamic Acid, Flumethasone, Flunisolide, Flunixin, Flunoxa profen, fluocinolone acetonide, fluocinonide, fluocinolone acetonide, fluocortin butyl, fluocortolone, fluoresone, fluorometholone, fluperolone, flupirtine, fluprednidene, fluprednisolone, fluproquazone, flurandrenolide, flurbipro Including Fen, Fluticasone, Formocortal and Phosphosal.

Further examples of anti-inflammatory agents are gentisic acid, graphenin, glucamethacin, glycol salicylate, guaiazulene, halcinonide, halobetasol, halomethasone, haloprednone, heroin, hydrocodone, hydrocortamate, hydrocortisone, hydrocortisone acetate, hydrocortisone succinate, hydrocortisone hemis Cucinate, hydrocortisone 21-lysinate, hydrocortisone cypionate, hydromorphone, hydroxypethidine, ibufenac, ibuprofen, ibuproxam, imidazole salicylic acid, indomethacin, indoprofen, isofezolac, isofulpredone, isofulpredone acetate, isoladol, isomethadone, isonixin, isoxepac, isoxicam, ketobemidone, ketoprofen, ketorolac, p-lactofenetide, lephetamine, levallorphan, levorphanol, levofenacyl-morphan, lofentanil, lonazolak, lornoxicam, loxoprofen, lysine acetylsalicylate, mazipredone, meclofenamic acid, medrysone, mefenamic acid, meloxicam, meperidine, meprednisone, meptazinol, mesalamine, metazocine, methadone, methotrimeprazine, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, methylprednisolone suleptnate , methiazic acid, metofolin, metopone, mofebutazone, mofezolac, mometasone, morazon, morphine, morphine hydrochloride, morphine sulfate, morpholine salicylate and myrofin.

Further examples of anti-inflammatory agents are nabumetone, nalbuphine, nalorphine, 1-naphthyl salicylate, naproxen, narcein, nefopam, nicomorphine, niphenazone, niflumic acid, nimesulide, 5′-nitro-2′-propoxyacetanilide, norlevorphanol, normethadone, normorphine, norpipanone, olsalazine, opium, oxaceprol, oxamethacin, oxaprozin, oxycodone, oxymorphone, oxyphenbutazone, papaveretam, paramethasone, paraniline, parsalmide, pentazocine, perisoxal, phenacetin, phenadoxone, phenazocine, phenazopyridine hydrochloride, phenocol, phenoperidine, phenopyrazone, phenomorphan, phenyl acetylsalicylate, phenylbutazone, phenyl salicylate, phenylamidol, piketoprofen, pimidine, pipebzone, piperylone, pirazolac, piritramide, piroxicam, Pirprofen, pranoprofen, prednicarbate, prednisolone, prednisone, prednivar, prednylidene, proglutacin, proheptadine, promedol, propacetamol, properidine, propyram, propoxyphene, propifenazone, proquazone, protidic acid, proxazole, lamiphenazone , remifentanil, Rimazolium Metilsulfate, salacetamide, salicin, salicylamide, salicylamido-O-acetic acid, salicylic acid, salicyl sulfate, salsalate, salverine, simethride, sufentanil, sulfasalazine , sulindac, superoxide dismutase, suprofen, suxibzone, talniflumate, tenidap, tenoxicam, terofenamate, tetrandrine, thiazolinobutazone, tiaprofenic acid, tiaramide, tilidine, tinoridine, tixocortol, tolfenamic acid, Including tolmetin, tramadol, triamcinolone, triamcinolone acetonide, tropecin, biminol, xenbucin, ximoprofen, zaltoprofen and zomepirac.

(F) Administration Patients are generally humans, but may be non-human mammals. Human patients are adults, such as patients between the ages of 19-25, 26-40, 41-55, 56-75, and 76 and older, eg, ages 0-2, 3-6, 7 and older. Includes pediatric patients, such as patients between the ages of 12 and 13-18.

The term "pharmaceutically acceptable" composition refers to non-toxic carriers or excipients with which the kallikrein inhibitors described herein can be administered to a patient. A carrier or excipient is selected to be compatible with the biological or pharmacological activity of the composition. The kallikrein inhibitors (and, in combination therapy, other therapeutic agents) described herein can be used to deliver inhibitory amounts of inhibitors and/or other therapeutic agents to patients, e.g., intravenously and Administration can be locally or systemically by any suitable means, including but not limited to systemic administration such as inhalation.

When administered parenterally, kallikrein inhibitors can be injected intravenously, intramuscularly, intraperitoneally, or subcutaneously. Subcutaneous injection and intravenous administration are preferred routes for parenteral administration. Local (intra-articular) injection is also useful.

Typically, compositions for administration by injection are solutions in sterile isotonic aqueous buffer (eg, sodium/potassium phosphate buffered saline). Other pharmaceutically acceptable carriers are sterile water, saline solutions and buffered saline (including buffers such as phosphate or acetate), alcohol, vegetable oils, polyethylene glycol, gelatin, lactose, amylose. , magnesium stearate, talc, silicic acid, paraffin, and the like. If necessary, the composition may also contain solubilizers, local anesthetics such as lidocaine to relieve pain at the injection site, preservatives, stabilizers, wetting agents, emulsifiers, salts, lubricants, etc. It can be included as long as it does not adversely react with the active ingredient. Likewise, the compositions may contain standard excipients such as pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral or intranasal administration which do not deleteriously react with the active ingredients. agent. Generally, the ingredients are presented separately or together in unit dosage form, e.g., as a lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule, sachet, or vial indicating the amount of active agent in units of activity. May be supplied either mixed. When the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade "water for injection" or saline. When the composition is administered by injection, a water for injection or saline container (eg, an ampoule or vial) can be supplied so that the active ingredients can be mixed prior to administration.

An exemplary formulation for subcutaneous administration of an isolated kallikrein inhibitor is a buffer (eg, histidine or phosphate buffer) and a cryoprotectant (eg, sucrose optionally containing a dextran such as dextran 40). or sucrose and mannitol), and storage and storage, as described in US Patent Application Publication No. 2007-0213275 (US Patent Application No. 11/716,278, filed March 9, 2007). It can be lyophilized for distribution.

In one embodiment, the kallikrein inhibitor is administered to the patient as an intravenous infusion according to any approved protocol. In other embodiments, the kallikrein inhibitor is administered to the patient as a subcutaneous bolus. In other embodiments, the kallikrein inhibitor is administered to the patient by intra-articular injection. Intravenous and intra-articular administration is typically performed by a health care professional in a clinical setting (e.g., hospital, emergency care, or doctor's office), whereas subcutaneous injection can be self-administered or administered by a medical practitioner. It can be administered by a professional.

Parameters that can be evaluated to determine the dose of a kallikrein inhibitor for systemic administration are described below with respect to DX-88 (a non-natural kallikrein inhibitor, SEQ ID NO:2). The total amount of circulating prekallikrein in plasma is reported to be approximately 500-600 nM (Silverberg et al., "The Contact System and Its Disorders," in Blood: Principles and Practice of Hematology, edited by Handin, R. et al., JB Lippincott Co., Philadelphia, 1995). About 520 nmole/L of DX-88 (DX88) can be used to stoichiometrically inhibit kallikrein if all prekallikrein is activated. An individual with 5 liters of plasma would require a dose of DX-88 of 2.6 micromolar, or approximately 18 mg, based on DX-88's molecular weight of 7,054 daltons. This was calculated as follows. The K _i for DX88 is 0.025 nM. For example, when it is desired to have a concentration of plasma kallikrein (PK) of 1 nM, the tight binding inhibitor formula below indicates that the concentration of free DX-88 is 12.0 nM. Therefore, the total amount of DX-88 required would be 499+12 or 511 nM.

If all of the prekallikrein is not activated, or if some kallikrein is inactivated by an endogenous inhibitor, such as the C1 esterase inhibitor (C1INH), the dose can be reduced proportionally. Thus, in certain embodiments, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 60 mg in a single dose or in one or more doses divided over a 24 hour period. , about 80 mg, about 120 mg, about 250 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 1000 mg of DX-88 can be administered to the subject. Consideration of several other factors, such as the patient's age, weight and severity of symptoms, can provide a more accurate estimate of the dose of DX-88 actually required.

In certain embodiments, the kallikrein inhibitor polypeptide is administered at a dose of about 1-500 mg/m ² , preferably about 1-250 mg/m ² , 1-100 mg/m ² .

(G) Devices and Kits Pharmaceutical compositions containing kallikrein inhibitors can be administered by medical devices. The device is designed for portability, room temperature storage, and ease of use so that it can be used in settings outside of a hospital or emergency room/emergency care facility (e.g., in the home or doctor's office by the patient or by the caregiver). It can be designed with such features. The device can include, for example, one or more housings for storing a pharmaceutical formulation comprising an isolated kallikrein inhibitor, and one or more unit doses of the agent or agents. can be configured to deliver

Intravenous administration can be by bolus or infusion using suitable injection or infusion devices (eg, catheters, infusion pumps, implants, etc.). Subcutaneous injection may be, for example, as an infusion using a catheter and infusion pump or implantable device. Many other devices, implants, delivery systems, and modules are also known.

When the kallikrein inhibitor is dispensed as a lyophilized powder, it must be reconstituted prior to use. Manual reconstitution (e.g., manual addition of diluent to the lyophilized formulation by injection through an injection port into the container containing the lyophilized formulation) can also be used, or the kallikrein inhibitor is Becton - may be provided in a device configured for automated reconstitution (eg, automated addition of diluents to lyophilized formulations), such as the Dickinson BD™ Liquid Dry Injector.

An isolated kallikrein inhibitor can be provided in a kit. In one embodiment, the kit comprises (a) a container comprising a composition comprising an isolated kallikrein inhibitor; May contain informational material related to use.

In some embodiments, the kit also contains another therapeutic agent. For example, a kit includes a first container containing a composition comprising an isolated kallikrein inhibitor and a second container containing another therapeutic agent. An isolated kallikrein inhibitor and other therapeutic agent may be supplied in the same container for use in methods in which the kallikrein inhibitor and therapeutic agent are administered as a single composition.

The informational material of the kit is not limited in its form. In one embodiment, the informational material may include information about compound manufacture, compound molecular weight, concentration, expiration date, batch or manufacturing location information, and the like. In one embodiment, the informational material is, e.g., in a suitable dose, dosage form or mode of administration (e.g., a dose, dosage form or mode of administration described herein) for treating a subject, It relates to a method of administering an isolated kallikrein inhibitor. The information may be supplied in a variety of formats including printed documents, computer readable materials, video or audio recordings, or information providing links or addresses to material material.

In addition to the isolated kallikrein inhibitor (and additional therapeutic agent, if present), the compositions in the kit may include other components such as solvents or buffers, stabilizers, or preservatives. obtain. The isolated kallikrein inhibitor (and additional therapeutic agent, if present) is preferably supplied in any form that is substantially pure and/or sterile, e.g., liquid, dried or lyophilized. obtain. When the agent is provided in a liquid solution, the liquid solution is preferably an aqueous solution. When the agent is provided in dry form, reconstitution is generally effected by addition of a suitable solvent. Solvents such as, for example, sterile water or buffers can optionally be provided in the kit.

A kit may include one or more containers for compositions containing compositions or agents. In some embodiments, the kit contains separate containers, dividers or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within single, undivided containers. For example, the composition may be contained in a bottle, vial or syringe having informational material in the form of a label applied thereon. In some embodiments, the kit comprises a plurality (eg, packs) of individual containers, each containing one or more unit dosage forms (eg, dosage forms described herein). The container may contain a combination unit dose, eg, a unit containing both the isolated kallikrein inhibitor and the other therapeutic agent, eg, in a desired ratio. For example, a kit may include multiple syringes, ampoules, foil packets, blister packs, or medical devices, eg, each containing a single combined unit dose. The containers of the kit may be airtight, waterproof (eg, impermeable to moisture changes or evaporation) and/or light-tight.

Kits optionally include a device suitable for administration of the composition, such as a syringe or other suitable delivery device. The device may be supplied prefilled with one or both of the agents or may be empty suitable for filling.
(Example)

EXAMPLES The following examples provide further illustration but are not limiting.

Example 1 Association of truncated high molecular weight kininogen (HMWK) with autoimmune diseases CD) and in plasma samples obtained from healthy subjects were measured using Western blot with LiCor detection, as described below.

(i) Sample Preparation Treated HMWK-deficient plasma was prepared by adding 10 μL of 10× anti-protease inhibitor cocktail to 90 μL of 100% HMWK-deficient plasma. The solution was allowed to sit for at least 30 minutes before use.

A 1:4 intermediate of single-chain HMWK was prepared by adding 5 μL of stock solution (1.61 mg/ml) to 15 μL of treated HMWK-deficient plasma. A 1:4 intermediate of double-stranded HMWK was prepared by adding 5 μL of stock solution (2.01 mg/ml) to 15 μL of treated HMWK-deficient plasma.

A 45 μg/mL treated HMWK-deficient control plasma solution was prepared with 3.35 μL of 1:4 single-chain HMWK intermediate and 2.69 μL of 1:4 It was prepared by adding a two-chain HMWK intermediate.

Each sample was diluted to 5% plasma (1:20) in 1×TBS by adding 5 μL of plasma sample to 95 μL of 1×TBS. Non-reduced samples were prepared by adding 5 μL of 4× sample buffer to 15 μL of 5% sample. Reduced samples were prepared by adding 5 μL of 4× sample buffer and 2 μL of 10× reducing agent to 13 μL of 5% sample.

All samples were heated at 95°C for 4 minutes using a heat block. Each sample was spun down briefly to remove any solution from the cap of the sample tube.

(ii) Gel loading, running and transfer Tris-acetate running buffer was prepared by adding 100 mL of 20× running buffer to 1900 mL of DI water. A volume of 4 μL of single-color protein marker was used as a control in all assays. A volume of 13 μL of non-reduced and reduced samples was added to the gel lanes of Gel 1. Gels were run at 125 volts for ˜2 hours.

The iBlot Filter Paper was placed in DI water and soaked for 5 minutes. The Anode Stack, Bottom, was unwrapped and placed copper side down on the blotting surface of the iBlot. Upon completion of gel run, the two gel cassettes were opened and the gel removed. Gels were mounted on transfer membranes. A presoaked filter paper was placed on top of the gel. The Cathode Stack, Top, was unsealed and placed copper side up on top of the filter paper. Air bubbles in the stack were slowly pushed out using a blotting roller. A disposable sponge was placed in the iBlot lid. The iBlot was switched on and program P0 was selected.

When the iBlot transfer was complete, the iBlot was opened and the sponge, cathode stack and gel were discarded. Each membrane was individually removed from the iBlot and placed in a plastic tray containing 20 mL of Odyssey Blocking Buffer. Membranes were incubated on a plate shaker for 1 hour at room temperature.

(iii) Western Blot Assay with LiCor Detection 1 μg/mL primary antibody solution was 57.14 μL mouse anti-LC HMWK, clone #11H05 (stock concentration 1.4 mg/mL). Blocking buffer was removed from the plastic tray. A volume of 20 mL primary antibody solution was added to each tray and the membranes were incubated on a plate shaker for 1 hour at room temperature. Goat anti-mouse IgG IRDye680 solution was prepared at 1:15,000 dilution by adding 5.33 μL of stock solution to 80 mL of Odyssey Blocker + 0.2% Tween-20. The primary antibody solution was removed from the plastic tray. The membrane was washed with 20 mL of 1×PBS+0.1% Tween-20 for 5 minutes, after which the wash was discarded. Washing was repeated 3 times for a total of 4 washes. A volume of 20 mL of goat anti-mouse IgG IRDye680 solution was added to each tray. The tray was covered with aluminum foil to protect the membrane and secondary antibody solution from light. Membranes were incubated on a plate shaker for 1 hour at room temperature. Secondary antibody solution was removed from the plastic tray. The membrane was washed with 20 mL of 1×PBS+0.1% Tween-20 for 5 minutes and the wash was discarded. Washing was repeated 3 times for a total of 4 washes. Membranes were washed with PBS and scanned on a LiCor Odyssey CLx. The membrane was covered with aluminum foil and dried overnight. The dried membrane was placed in a protective coversheet and stored for later use.

(iv) Results Control treated HMWK-deficient plasma spiked with 45 μg/mL single- and double-chain HMWK produced bands around 120 kDa and 95 kDa, as expected. Normal human plasma produced bands around 120 kDa and 100 kDa, with 21.3% of HMWK cleaved. Patient samples containing an anti-protease inhibitor cocktail yielded dramatically different results than patient samples collected in sodium citrate. Patient samples taken with antiprotease inhibitors contained significantly more single-chain HMWK than reduced double-chain HMWK. This result indicates that the addition of an anti-protease inhibitor cocktail is necessary to prevent cleavage of HMWK after harvesting.

Plasma samples from multiple UC, CD, RA and HAE patients were examined in this study. Control samples and normal human plasma gave the expected results. HAE attack samples contained more truncated HMWK than HAE reference samples. As shown in Table 1 below and in Figure 1, the majority of samples from patients with autoimmune diseases, especially rheumatoid arthritis samples, yielded only the 46 kDa band, with concentrations of truncated HMWK ranging from RA, UC and CD. It has been shown to be associated with autoimmune diseases such as More specifically, exacerbations of RA were associated with high amounts of truncated HMWK, Crohn's disease was found to have moderate amounts of truncated HMWK, and ulcerative colitis was associated with moderate amounts of truncated HMWK. It was found to have a large amount of truncated HMWK.

Taken together, the results of this study demonstrate plasma contact activation of autoimmune diseases such as CD, UC and RA. Accordingly, agents that inhibit contact activation, such as inhibitors of pKal (eg, those described herein), may be effective in treating such diseases. Furthermore, the results of this study indicate that the concentration of truncated HMWKs is a reliable biomarker for identifying patients with or at risk of autoimmune diseases such as CD, UC and RA, and/or , that it can serve as a biomarker to determine the efficacy of treatments for such autoimmune diseases.

The contents of all cited references, including references, issued patents, published or unpublished patent applications, and those listed below, cited throughout this application, are for the purposes of reference herein or The subject matter is hereby expressly incorporated by reference in its entirety. In case of conflict, the present application, including any definitions herein, will control.

Equivalents and Ranges Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. . The scope of the present disclosure is not intended to be limited to the foregoing description, but rather is set forth in the appended claims.

In the claims, articles such as "a", "an" and "the" may mean one or more than one unless the context clearly indicates otherwise or to the contrary. . A claim or statement containing "or" between one or more members of a group, in a given product or process, unless the context clearly indicates to the contrary or otherwise: One or more or all of the members of a group are considered satisfied if present, used, or otherwise associated. The present disclosure includes embodiments in which exactly one member of the group is present, used or otherwise associated with a given product or process. The disclosure includes embodiments in which one or more of the group or all members of the group are present, used or otherwise associated in a given product or process.

Furthermore, the present disclosure covers all modifications, combinations and Including substitutions. For example, any claim that is dependent on any other claim may be modified to include one or more limitations found in any other claim that is dependent on the same base claim. When elements are present as enumerations, eg, in Markush group form, subgroups of each element are also disclosed, and any element can be removed from the group. Generally, when this disclosure or aspects of this disclosure are presented as including certain elements and/or features, certain embodiments of this disclosure or aspects of this disclosure consist of such elements and/or features. or consist essentially of them. For the sake of simplicity, such embodiments are not specifically described in such language herein. The terms "comprising" and "containing" are intended to be open-ended and allow the inclusion of additional elements or steps. When a range is given, the endpoints are included. Further, unless otherwise indicated or obvious from the context and the understanding of one of ordinary skill in the art, values expressed as ranges are units of units at the lower end of the range, unless the context clearly dictates otherwise. Any specific value or subrange within the ranges stated in the other embodiments of this disclosure, up to a tenth, can be taken.

This application references various issued patents, published patent applications, journal articles and other publications, all of which are incorporated herein by reference. In the event of a conflict between any of the incorporated references and this specification, the specification will control. Moreover, any particular implementation of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. As such implementations are considered known to those skilled in the art, they may be excluded even if the exclusion is not expressly described herein. Any particular implementation of the present disclosure may be excluded from any claim for any reason, whether related to the existence of prior art or not.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the embodiments described herein is not intended to limit the above description, but rather is set forth in the appended claims. Those skilled in the art will recognize that various changes and modifications may be made to this description without departing from the spirit or scope of the disclosure as set forth in the claims below.

Claims (12)

1. A composition for use in treating an autoimmune disease in a subject comprising a plasma kallikrein (pKal) inhibitor , comprising:
said subject has an elevated percent truncated high molecular weight kininogen (HMWK) compared to a control sample;
said pKal inhibitor is a Kunitz domain polypeptide or an antibody that binds to pKal;
The percent truncated HMWK is measured in an assay containing a binding agent specific for truncated HMWK ,
the sample is a biological sample that is a serum sample or a plasma sample ;
Composition. 2. The composition of claim 1, wherein the autoimmune disease is rheumatoid arthritis (RA), Crohn's disease (CD) or ulcerative colitis (UC). 3. The composition of claim 1 or 2, wherein said pKal inhibitor is DX-2930 or DX-88. The composition of any one of claims 1-3, wherein said assay involves providing one or more biological samples of said subject. The composition of any one of claims 1-4, further comprising an anti-inflammatory agent. 6. The composition of claim 4 or 5, wherein at least one of said biological samples comprises one or more protease inhibitors added to said sample after its collection. The composition of any one of claims 1-6, wherein the binding agent is an antibody that binds to truncated HMWK. The composition of any one of claims 1-7, wherein the assay is an enzyme-linked immunosorbent assay (ELISA) or an immunoblot assay. The composition of any one of claims 1-8, wherein said assay is a Western blot assay. The composition of any one of claims 1-9, wherein the subject is a human subject. The composition of any one of claims 1-10, wherein the control sample is a sample from a healthy subject. A composition according to any one of claims 5 to 11, wherein said anti-inflammatory agent is a steroid or non-steroidal anti-inflammatory drug (NSAID).

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